Beta lactamase as biomarker for the specific detection of tuberculosis-complex bacteria

ABSTRACT

The present disclosure provides methods, reagents, systems, and devices that target β lactamase as a biomarker for the sensitive and specific detection of tuberculosis-complex bacteria. Specifically, the present disclosure relates to methods and compositions for the detection of specific β-lactamase protein and nucleic acid sequences to indicate the presence of tuberculosis-complex bacteria.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/038,736 filed Aug. 18, 2014, and U.S. Provisional Application No.62/134,332, filed Mar. 17, 2015, each of which is incorporated herein byreference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

The sequence listing associated with this application is provided intext format in lieu of a paper copy and is hereby incorporated byreference into the specification. The name of the text file containingthe sequence listing is 54434_SEQ_ST25.txt The text file is 16 KB; wascreated on Aug. 17, 2015; and is being submitted via EFS-Web with thefiling of the specification.

BACKGROUND

Tuberculosis (TB) is one of the most important infectious diseases inhumans and animals worldwide. The World Health Organization currentlyestimates that roughly one-third of the world's population is infectedwith tuberculosis (TB), caused by the Mycobacterium tuberculosis (Mtb).In the year 2011 alone, 8.7 million people fell ill with TB and another1.4 million died. While the risk of developing symptoms from the latentcondition is only 10%, this number increases greatly if the individualis also infected with an immune compromising disease such as HIV. TB isa treatable and curable disease, typically combated with a six-monthcourse of antimicrobial drugs, and the use of these treatments hassignificantly decreased the mortality rate for TB over the last quartercentury. Despite this, multi-drug resistant TB strains generate concernamong medical experts and demand the need for the development of newantimicrobial strategies. One important component to treatmentstrategies is the implementation of effective and accurate diagnosis andtracking of infections, including latent infections. Such diagnosticstrategies could dramatically enhance the ability to detect infectionand potentially prevent transmission, thus reducing the overallincidence of TB.

Early diagnosis is critical to the prevention and control oftuberculosis due to its airborne transmission. Standard diagnosticmethods, such as an acid-fast stain on smears from sputum, do not becomepositive until after transmission can occur, allowing spread of disease.Culture-based techniques are more sensitive, but take weeks to obtainresults, due to the extremely slow growth rate of TB bacteria. Thus,clinical diagnosis and disease control would be greatly facilitated bymethods that can detect tubercle bacteria in a sensitive, rapid,specific and quantitative manner during disease.

In addition to this, the current vaccine, Mycobacterium bovis BacillusCalmette Guerin (BCG), displays variable efficacy (0-80%) depending onthe population being vaccinated. Currently, researchers typically relyon animal studies to help assess the effectiveness of new therapeuticagents. These studies employ sacrifice at discrete time points, tissuehomogenization, and colony growth. These factors combine to greatlylimit temporal and spatial resolution of the bacteria in tissue. Thus,the development of an experimental technique that could provide rapidfeedback regarding the efficacy of a therapeutic agent in an animalmodel of a respiratory infection could greatly benefit the developmentof such vaccines.

Despite the advances in the development of diagnostic techniques for thediagnosis and monitoring of TB infections, a need remains for sensitive,rapid, and specific diagnostic reagents and methods that facilitate therapid detection of tuberculosis. The present invention seeks to fulfillthis need and provides further related advantages.

SUMMARY

This summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This summary is not intended to identify key features ofthe claimed subject matter, nor is it intended to be used as an aid indetermining the scope of the claimed subject matter.

In one aspect, the present disclosure provides an affinity reagent thatspecifically binds to a β-lactamase (BlaC), or a portion thereof, of atuberculosis-complex bacteria.

In some embodiments, the BlaC has an amino acid sequence with at least95% identity to the sequence set forth in SEQ ID NO:2. In someembodiments, the BlaC has an amino acid sequence set forth in SEQ IDNO:2. In some embodiments, the affinity reagent is capable ofspecifically binding to a portion of BlaC.

In some embodiments, the affinity reagent is an antibody, an enzymesubstrate, a modified enzyme substrate, and the like. In someembodiments, the affinity reagent is a polyclonal or monoclonalantibody.

In some embodiments, the tuberculosis-complex bacteria are from one ormore of the species selected from the group consisting of: Mycobacteriumtuberculosis, Mycobacterium bovis, Mycobacterium bovis-BacillusCalmete-Guarin (BCG), Mycobacterium africanum, Mycobacterium microti,Mycobacterium canettii, Mycobacterium pinnipedii, and Mycobacteriummungi.

In another aspect, the disclosure provides a method of detecting thepresence of tuberculosis-complex bacteria in a biological sample, themethod comprising 1) contacting the sample with an affinity reagent thatspecifically binds to a β-lactamase (BlaC) of a tuberculosis-complexbacterium, and 2) detecting the formation of a complex between the BlaCand the affinity reagent. The formation of a complex is indicative ofthe presence of tuberculosis-complex bacteria in the sample.

In some embodiments, the BlaC has an amino acid sequence with at least95% identity to the sequence set forth in SEQ ID NO:2. In someembodiments, the BlaC has an amino acid sequence set forth in SEQ IDNO:2. In some embodiments, the affinity reagent is capable ofspecifically binding to a portion of the BlaC.

In some embodiments, the affinity reagent comprises a detectable label.In some embodiments, the method further comprises contacting thebiological sample with an immobilized BlaC protein or affinity reagent.In some embodiments, the affinity reagent is attached to a substrate. Insome embodiments, the formation of a complex between the BlaC and theaffinity reagent is detected by further contacting the complex with asecond affinity reagent that contains a detectable label and thatspecifically binds to the complex.

In some embodiments, the biological sample is obtained from a subjectsuspected of having tuberculosis-complex bacteria, and wherein thepresence of tuberculosis-complex bacteria in the biological sample isindicative of a tuberculosis infection in the subject. In someembodiments, the biological sample comprises blood, serum, sputum,saliva, breath, feces, urine, spinal fluid, mucus, tissue sample, andthe like. In some embodiments, the subject is a mammal, such as a human.

In another aspect, the disclosure provides a method of detecting thepresence of tuberculosis-complex bacteria in a biological sampleobtained from a subject, the method comprising determining the presenceor amount of anti-β-lactamase (BlaC) antibody in a biological sample,wherein the presence or amount of anti-BlaC antibody in the biologicalsample is indicative of the presence of tuberculosis-complex bacteria inthe subject.

In some embodiments, the presence of anti-BlaC antibody in thebiological sample is determined by an assay comprising the followingsteps:

(a) contacting the biological sample with at least one polypeptide withan amino acid sequence that has at least 90% sequence identity to anysix or more contiguous amino acids of SEQ ID NO:2; and

(b) detecting the formation of a complex between the antibody in thesample and the polypeptide.

In some embodiments, the method further comprises comparing thedetermined amount of anti-BlaC antibody to a reference standard, wherean amount of anti-BlaC antibody detected in the biological samplegreater than the reference standard is indicative of the presence oftuberculosis-complex bacteria in the subject. In some embodiments, thereference standard is an analogous biological sample from a subject thatdoes not have tuberculosis-complex bacteria.

In another aspect, the present disclosure provides a method fordetecting the presence of tuberculosis-complex bacteria in a testsample, the method comprising:

(a) contacting the sample with a polynucleotide probe with a detectablelabel capable of specifically hybridizing to a target region of anucleic acid molecule that encodes a β-lactamase (BlaC) with an aminoacid sequence set forth in SEQ ID NO:2, and

(b) detecting the hybridization of the probe to the nucleic acidmolecule encoding BlaC, wherein detected hybridization is indicative ofthe presence of tuberculosis-complex bacteria in the test sample.

In some embodiments, the polynucleotide probe has a polynucleotidesequence selected from the group consisting of SEQ ID NO:3, 4, 5, 6, 7,8, 9, 10, and 11.

In some embodiments, the method further comprises contacting the samplewith a forward polynucleotide primer and a reverse primer to form areaction mixture, wherein each primer capable of specificallyhybridizing to a different portion of the target region, and subjectingthe reaction mixture to amplification conditions suitable to amplify atleast a portion of the target region. In some embodiments, the forwardprimer has a polynucleotide sequence selected from the group consistingof SEQ ID NO:12, 13, 14, 15, 16, 17, 18, 19, and 20. In someembodiments, the reverse primer has a polynucleotide sequence selectedfrom the group consisting of SEQ ID NO:21, 22, 23, 24, 25, 26, 27, 28,and 29. In some embodiments, the test sample is obtained from a subjectsuspected of having tuberculosis-complex bacteria, and wherein thepresence of tuberculosis-complex bacteria in the biological sample isindicative of a tuberculosis infection in the subject.

In another aspect, the present disclosure provides a method fordetermining the presence of tuberculosis complex bacteria in a testsample, the method comprising the steps of:

(a) contacting the test sample with a composition comprising at leastone primer pair comprising a forward and reverse primer capable ofspecifically hybridizing to a target region of tuberculosis complex blaCgene, to form a reaction mixture;

(b) subjecting said reaction mixture to amplification conditionssuitable to amplify at least a portion of the target region; and

(c) detecting amplification of the at least a portion of the targetregion, wherein amplification of the at least a portion of the targetregion is indicative of the presence of tuberculosis complex bacteria ina test sample.

In some embodiments, the primer pair is selected from the groupconsisting of SEQ ID NOS:12 and 21, SEQ ID NOS:13 and 22, SEQ ID NOS:14and 23, SEQ ID NOS:15 and 24, SEQ ID NOS:16 and 25, SEQ ID NOS:17 and26, SEQ ID NOS:18 and 27, SEQ ID NOS:19 and 28, and SEQ ID NOS:20 and29.

In another aspect, the present disclosure provides a method formonitoring the efficacy of treatment of a tuberculosis infection,comprising:

(a) determining the presence or amount of BlaC protein, nucleic acidencoding BlaC protein, or anti-BlaC antibodies in a biological sampleobtained from a subject receiving treatment for tuberculosis; and

(b) comparing the amount of BlaC protein, nucleic acid encoding BlaCprotein, or anti-BlaC antibodies in the biological sample as determinedin step (a) to a reference standard, thereby determining the efficacy oftreatment.

In some embodiments, the reference standard in step (b) is the amount ofBlaC protein, nucleic acid encoding BlaC protein, or anti-BlaCantibodies determined in an analogous biological sample obtained fromthe subject at or after diagnosis with the tuberculosis infection butprior to the obtaining of the biological sample from the subject in step(a), whereby a lower amount of anti-BlaC antibodies in the biologicalsample determined in step (a) compared to the biological sample in step(b) is indicative of a positive response to the treatment.

In another aspect, the present disclosure provides an isolatedpolynucleic acid molecule comprising a detectable label, wherein thepolynucleic acid molecule has a polynucleotide sequence set forth in asequence selected from the group consisting of SEQ ID NOS:3-29, orhomologs thereof.

In another aspect, the present disclosure provides a kit comprising inone or more containers DNA polymerase enzyme, deoxynucleosidetriphosphates, buffer solution, and an isolated polynucleic acidmolecule comprising a detectable label, as described herein. In someembodiments, the kit comprises a polynucleotide primer pair selectedfrom the group consisting of SEQ ID NOS:12 and 21, SEQ ID NOS:13 and 22,SEQ ID NOS:14 and 23, SEQ ID NOS:15 and 24, SEQ ID NOS:16 and 25, SEQ IDNOS:17 and 26, SEQ ID NOS:18 and 27, SEQ ID NOS:19 and 28, and SEQ IDNOS:20 and 29, or homologs thereof.

DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1A graphically illustrates the hydrolysis of nitrocefin by BlaCover time. Hydrolyzed nitrocefin products demonstrate a maximumabsorption at 485 nm. The formation of the hydrolyzed product wasmonitored by absorbance at 486 nm (A₄₈₆) as a function of time usingdifference concentrations of purified BlaC enzyme (concentrationsindicated with arrows).

FIG. 1B graphically illustrates the enzyme activity of BlaC as afunction of enzyme concentration. The observed rate of change at A₄₈₆was converted to units of enzyme activity (μM product formed per minute)using the molar extinction coefficient of the hydrolyzed product of BlaCβ-lactamase activity, which exhibits maximum absorption at 485 nm.

FIG. 2 graphically illustrates a standard plot of BlaC in sputum asdetermined using ELISA. Absorbance at 415 nm demonstrated a linearincrease with the increase in BlaC concentration in sputum. The standarddeviations and p-values are indicated.

FIG. 3 graphically illustrates the detection of BlaC in M. bovis (BCG)bacterial dilutions in sputum using ELISA. The absorbance values (at 415nm) for various CFU/well are indicated at the top of the bars.

FIG. 4 is a force tree illustrating the phylogenetic relationships ofMycobacterium species based on the nucleotide sequences of β-lactamase.The sequences were compared between species in the TB-complex, and themost closely related Mycobacterium species that is not in theTB-complex, i.e., Mycobacterium marinum. The phylogenetic tree wasgenerated by ClustalW2 (EMBL) using the nearest neighbor method anddemonstrates that blaC is highly conserved in the TB-complex.Mycobacterium marinum, which is not a member of the complex, isseparated from the complex by two nodes (phylogenetic distance).

FIG. 5 illustrates an amino acid sequence alignment of BlaC protein fromseveral TB-complex species. The BlaC protein in the TB-complex is highlyconserved (100% identity). The three signature motifs are shown in green(“Motif I”), red (“Motif II”) and blue (“Motif III”). The illustratedamino acid sequence for M. tuberculosis BlaC protein, and the identicalconsensus sequence for the BlaC protein of the indicated TB-complexbacteria, is set forth herein as SEQ ID NO:2.

FIG. 6 illustrates an amino acid sequence alignment of BlaC of M.tuberculosis with that of other pathogenic bacteria that are not part ofthe TB complex. The BlaC protein of M. tuberculosis displays lowsimilarity to the other β-lactamases. Sequence identifiers for eachamino acid sequence are indicated in the figure. The sequences for thethree signature motifs (i.e., I, II, and III) of the M. tuberculosisBlaC sequence are indicated.

FIG. 7 illustrates a sequence alignment of the β-lactamase gene fromvarious relevant pathogenic bacteria, including M. tuberculosis. Thealignment demonstrates that the blaC gene of M. tuberculosis (set forthherein as SEQ ID NO: 1) has low similarity with other β-lactamase genes.

FIG. 8 graphically illustrates the amplification plots of representative10 ng (upper line after round 35) and 50 ng (lower line after round 35)samples of template.

FIG. 9 provides illustrative test strips run using the wet method usingGoat anti-BlaC as an immobilized capture antibody and RabMab 22-12 as agold conjugated detection antibody. From left to right: running bufferas negative control, 50 ng/ml, and 3 ng/ml BlaC. 0.5 mg/ml Donkeyanti-Goat, Goat anti-Rabbit, Goat anti-Mouse, or combinations of thesecontrol capture antibodies were striped on a control line (CL) toindicate that binding conditions were proper.

FIG. 10 provides illustrative test strips run using the wet method usingRabMab 20-8 as an immobilized capture antibody and RabMab 27-11 as agold conjugated detection antibody. From left to right: running bufferas negative control, 50 ng/ml, and 3 ng/ml BlaC. 0.5 mg/ml Donkeyanti-Goat, Goat anti-Rabbit, Goat anti-Mouse, or combinations of thesecontrol capture antibodies were striped on a control line (CL) toindicate that binding conditions were proper.

FIG. 11A graphically illustrates the Axxin readings of BlaC asdetermined from 5 μm filtered and spiked sputum.

FIG. 11B graphically illustrates the Axxin readings of BlaC asdetermined from 0.2 μm filtered and spiked sputum.

FIG. 12 graphically illustrates the Axxin readings of BlaC present insaliva sample.

FIG. 13 graphically illustrates an exemplary primer pair, namely Mouseanti-BlaC H1 mAb as capture antibody and purified anti-BlaC rabbit(polyclonal) IgG as detection antibody, that successfully detected thepresence of BlaC from the Sauton's medium supernatant from a culture ofvarying densities of Mycobacterium tuberculosis. Samples of PBS bufferand Sauton's medium only were used as controls.

FIG. 14 graphically illustrates the lack of cross reactivity between thedisclosed anti-BlaC reagents and related, non TB-complex β lactamaseproteins.

FIG. 15 graphically illustrates the amount of non TB-complex β lactamaseproteins required to produce an equivalent signal to 5 ng/mL BlaC.

DETAILED DESCRIPTION

The present disclosure generally relates to the specific detection oftuberculosis-complex bacteria using β-lactamase as a biomarker.Specifically, the present disclosure relates to methods and compositionsfor the detection of specific β-lactamase protein and nucleic acidsequences to indicate the presence of tuberculosis-complex bacteria.

As used herein, the term “tuberculosis-complex bacteria” refers tobacteria from any species of a closely related group of Mycobacteriumthat can cause tuberculosis (TB). The tuberculosis complex, orTB-complex, can include bacteria from at least the following recognizedspecies: Mycobacterium tuberculosis (Mtb), Mycobacterium afiicanum,Mycobacterium bovis, Mycobacterium bovis Bacillus Calmette Guerin (BCG),Mycobacterium microti, Mycobacterium caneutii, Mycobacterium pinnipedii,and Mycobacterium mungi. These and other mycobacteria species aregenerally considered to be Gram-positive. However, because they lack thetypical outer cell membrane, they do not retain the crystal violet Gramstain very well. Because they can be imaged with an acid-fast technique,they are often referred to as acid-fast Gram positive.

As used herein, the term “tuberculosis” (“TB”) refers to the common, andoften lethal, disease. TB is currently in the top-ten causes of deathfor humans worldwide. A TB infection is generally classified as beingeither latent or active. Latent TB occurs when the bacteria are presentin the body, but in an inactive state. The inactivity might be theresult of immune mechanisms that prevent bacterial growth and spreading.For example, scar tissue or fibrosis can form around the TB bacteria toisolate the bacteria and to prevent the infection from spreading. Latentinfections typically present no symptoms and are not contagious. Incontrast, active TB is contagious and is the condition that isassociated with various symptoms. Active TB can affect almost any tissueor organ in the body, but the most common site of disease is in thelungs. With reference to lung infections, when the bacteria multiply,they can cause pneumonia along with chest pain, coughing up blood, and aprolonged cough. TB is infectious because TB-complex bacteria can bepassed through the air in water droplets when subjects with activeinfections cough, sneeze, or otherwise eject fluids into the air.Additionally, lymph nodes near the heart and lungs often become enlargedduring infection. The disease can progress, causing pneumonia and damageto kidneys, bones, and the meninges that line the spinal cord and brain.Classic symptoms of active TB infections include unexplained weightloss, tiredness, fatigue, shortness of breath, fever, night sweats,chills, and a loss of appetite. Symptoms specific to the lungs includecoughing that lasts for 3 or more weeks, coughing up blood, chest pain,and pain with breathing or coughing. Various antibiotics are used totreat TB infections, depending on whether the infection is latent oractive. Because treatments tend to require prolonged administration ofantibiotics, many patients do not complete the entire course, which canlead to antibiotic resistance and disease management challenges.

As used herein, the term “β-lactamase” refers to an enzyme produced byvarious bacteria known to confer resistance to certain β-lactamantibiotics, such as penicillins, cephamycins, and some carbapenems.β-lactamases are known to be secreted, and in some gram-negativebacteria an increase in secreted levels occurs when antibiotics are inthe environment. β-lactamases are classified based on the amino acid(and encoding nucleic acid) sequences of the enzymes. Currently, fourclasses (A-D) have been characterized. TB-complex bacteria naturallyexpress β-lactamase (BlaC) that belongs to “class A” of the β-lactamasefamily.

The present disclosure is based, in part, on the discovery that unlikevarious other bacteria, the blaC gene is constitutively expressed athigh levels by TB-complex bacteria under almost all growth conditions.The BlaC protein specifically localizes on the surface of the bacteriaand is also constitutively secreted under various growth conditions. Asdescribed in more detail below, comparative analyses revealed numerousdifferences between the BlaC amino acid sequence, and their encodingnucleic acid blaC sequence, of TB-complex bacteria and the β-lactamasesequences from other, non-TB-complex bacteria (i.e., other bacteria,even including mycobacterial species that do not cause tuberculosis inhumans or animals). Thus, reagents and methods that specifically detectBlaC protein or blaC nucleic acid sequences from the TB-complex bacteriahave utility for the specific and sensitive detection and diagnosis ofTB-complex bacteria.

Accordingly, in one aspect, the present disclosure provides an affinityreagent that specifically binds to a β-lactamase (BlaC) of a TB-complexbacterium.

As used herein, the term “specifically binds” refers to the ability ofthe affinity reagent to bind to the BlaC protein, without significantbinding to other molecules, such as β-lactamase (Bla) protein ofnon-TB-complex bacteria, under standard conditions known in the art. Theaffinity reagent can bind to other peptides, polypeptides or proteins,but with lower affinity as determined by, e.g., immunoassays, BIAcore,or other assays known in the art. Affinity reagents preferably do notcross-react with other proteins, such as β-lactamase (Bla) protein ofnon-TB-complex bacteria. For example, the affinity reagent preferablybinds to the BlaC protein in a manner that is detectable over backgroundbinding.

As used herein, the term “affinity reagent” refers to any moleculehaving an ability to bind to a specific molecule with a specificaffinity (i.e., detectable over background). More specifically, in thecontext of the present disclosure, the term generally refers to amolecule having the ability to specifically bind to a β-lactamase (BlaC)of a TB-complex bacterium. The affinity reagent can be an antibody, anenzyme substrate, a modified enzyme substrate, and the like.

In some embodiments, the affinity reagent is an antibody. As usedherein, the term “antibody” encompasses antibodies and antibodyfragments thereof, derived from any antibody-producing mammal (e.g.,mouse, rat, rabbit, and primate including human), that specifically bindto a polypeptide target of interest, such as BlaC, or portions thereof.Exemplary antibodies include polyclonal, monoclonal and recombinantantibodies; multispecific antibodies (e.g., bispecific antibodies);humanized antibodies; murine antibodies; chimeric, mouse-human,mouse-primate, primate-human monoclonal antibodies; and anti-idiotypeantibodies, and may be any intact molecule or fragment thereof.

An antibody fragment is a portion derived from or related to afull-length antibody, preferably including the antigen binding orvariable region thereof. Illustrative examples of antibody fragmentsuseful in the present disclosure include Fab, Fab′, F(ab)₂, F(ab′)₂ andFv fragments, scFv fragments, diabodies, linear antibodies, single-chainantibody molecules, multispecific antibodies formed from antibodyfragments, and the like. A “single-chain Fv” or “scFv” antibody fragmentcomprises the V_(H) and V_(L) domains of an antibody, wherein thesedomains are present in a single polypeptide chain. The Fv polypeptidecan further comprise a polypeptide linker between the V_(H) and V_(L)domains, which enables the scFv to form the desired structure forantigen binding. Antibody fragments can be produced recombinantly, orthrough enzymatic digestion.

Antibodies can be further modified to suit various uses. For example, a“chimeric antibody” is a recombinant protein that contains the variabledomains and complementarity-determining regions (CDRs) derived from anon-human species (e.g., rodent) antibody, while the remainder of theantibody molecule is derived from a human antibody. A “humanizedantibody” is a chimeric antibody that comprises a minimal sequence thatconforms to specific complementarity-determining regions derived fromnon-human immunoglobulin that is transplanted into a human antibodyframework. Humanized antibodies are typically recombinant proteins inwhich only the antibody complementarity-determining regions (CDRs) areof non-human origin.

Production of antibodies can be accomplished using any techniquecommonly known in the art. For example, the production of a polyclonalantibody described herein can be generated by administering an immunogencontaining the target antigen, such as BlaC or a fragment thereof, to anantibody-producing animal. For example, the target antigen can beadministered to a mammal (e.g., a rat, a mouse, a rabbit, a chicken,cattle, a monkey, a pig, a horse, a sheep, a goat, a dog, a cat, aguinea pig, a hamster) or a bird (e.g., a chicken) so as to induceproduction of a serum containing an antigen-specific polyclonalantibody. The target antigen can be administered in combination withother components known to facilitate induction of a B-cell response,such as any appropriate adjuvant known in the art. For example, asdescribed below in more detail below, recombinant BlaC was administeredto rabbits to obtain a population of polyclonal antibodies that weredemonstrated as useful reagents in the detection of BlaC (andTB-bacteria expressing BlaC) in sputum. Furthermore, the polyclonalantibody reagent can be further processed to remove any antibody membersthat have unacceptable affinity for non-BlaC antigen. For example, toensure specificity of a polyclonal antibody reagent for BlaC over anyparticular β-lactamase protein(s) from non-TB complex bacteria, thepolyclonal antibody can be contacted to immobilized β-lactamaseprotein(s) under conditions that permit binding. In this way, antibodiesfrom the polyclonal reagent that bind can be removed from the reagentwhen the unbound antibodies are collected. The resulting polyclonalantibody reagent will exhibit enhanced specificity for BlaC and areuseful for detection of BlaC and/or TB-complex bacteria. Many approachesfor adsorption of polyclonal antibody reagents to reducecross-reactivity exist, are familiar to persons of ordinary skill in theart, and are encompassed by the present disclosure.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981), incorporated herein byreference in their entireties. The term “monoclonal antibody” refers toan antibody that is derived from a single clone, including anyeukaryotic, prokaryotic, or phage clone, and not the method by which itis produced. Methods for producing and screening for specific antibodiesusing hybridoma technology are routine and well known in the art. Anillustrative strategy for producing monoclonal antibodies is describedbelow.

Antibody fragments that recognize specific epitopes can be generated byany technique known to those of skill in the art. For example, Fab andF(ab′)₂ fragments of the invention can be produced by proteolyticcleavage of immunoglobulin molecules, using enzymes such as papain (toproduce Fab fragments) or pepsin (to produce F(ab′)₂ fragments). F(ab′)₂fragments contain the variable region, the light chain constant regionand the CHI domain of the heavy chain. Further, the antibodies of thepresent invention can also be generated using various phage displaymethods known in the art.

In some embodiments, the BlaC protein comprises at least 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or moreconsecutive amino acid residues with a sequence that is at least 95%,96%, 97%, 98%, or 99% identical to a sequence fragment of the amino acidsequence set forth in SEQ ID NO:2. In some embodiments, the BlaC proteinhas an amino acid sequence that is at least 95%, 96%, 97%, 98%, or 99%identical to the amino acid sequence set forth in SEQ ID NO:2. As usedherein, the terms indicating “percent identity” or “percent identical”refer to the percentage of amino acid residues in a polypeptide sequencethat are identical with the nucleic acid sequence or amino acid sequenceof a specified molecule, after aligning the sequences to achieve themaximum percent identity. For example, the Vector NTI Advance™ 9.0 maybe used for sequence alignment. An exemplary BlaC is provided in theGenBank database under accession number WP_003410677.1. In someembodiments, the BlaC protein comprises an amino acid sequence set forthin SEQ ID NO:2, or a fragment thereof with at least 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more aminoacids. In some embodiments, the fragment comprises amino acidscorresponding to amino acids within positions 75-87 of SEQ ID NO:2,within amino acids at positions 142 to 144 of SEQ ID NO:2, and/or withinamino acids at positions 249 to 252 of SEQ ID NO:2. Such regions areknown to participate in structural motifs that are important forenzymatic activity (see FIG. 5).

In some embodiments, the affinity reagent is capable of specificallybinding to the BlaC protein or any antigenic fragment or portionthereof. Antigenic fragments can be predicted using known methods.

In some embodiments, the affinity reagent is detectably labeled tofacilitate detection of complex formation between the target BlaCprotein, or a fragment thereof, and the affinity reagent. The detectablelabels are described in more detail below.

The affinity reagents described above are useful for the specificcapture and/or detection of BlaC protein to indicate the presence ofTB-complex bacteria. Accordingly, in one aspect, the present disclosureprovides a method of detecting the presence of tuberculosis-complexbacteria in a sample, comprising contacting the sample with an affinityreagent described herein. As described, the affinity reagentspecifically binds to a BlaC protein of a TB-complex bacterium, or afragment thereof. The method further comprises detecting the formationof a complex between the BlaC protein and the affinity reagent, whereinthe formation of a complex is indicative of the presence of tuberculosiscomplex bacteria in the sample.

According to this aspect, the detection of a complex between the BlaCprotein and the affinity reagent can be performed according to any knownassay format for this purpose. Such assays for the detection and/orquantification of the BlaC protein typically involve incubation of thesample that potentially contains the target BlaC protein with theaffinity reagent, and detection via the formation of a complex betweenthe affinity reagent and the target BlaC protein. In variousembodiments, either the components of the biological sample (includingthe target BlaC protein) or the affinity reagents are immobilized. Insome embodiments, either the affinity reagent or some component oftarget BlaC protein is modified in a manner that it provides adetectable signal. Exemplary techniques include immunoassays, such as insitu hybridization, western blots, immunoprecipitation followed bySDS-PAGE electrophoresis, immunocytochemistry, ELISA, and the like, someof which are described in more detail below.

Immunoassays encompassed by the present disclosure can be organized in anumber of different formats recognized in the art. For example, incompetitive immunoassays, unlabeled analyte from a biological samplecompetes with a labeled version of the analyte, such as BlaC protein,for binding to an affinity reagent. The amount of labeled, unboundanalyte is then measured. The more unlabeled analyte in the biologicalsample results in more labeled analyte that is displaced or competed offof the affinity reagent. Thus, the amount of labeled, unbound analytethat can be rinsed away is proportional to the amount of unlabeledanalyte present in the biological sample. In a variation of thisembodiment, the amount of labeled, bound analyte is measured, which isinversely proportional to the amount of unlabeled analyte present in thebiological sample. In some embodiments, the affinity reagent isimmobilized to facilitate the rinsing of the reagent, without losing thebound analytes.

Some formats are non-competitive. In one example, in situ hybridizationutilizes a combination of immunofluorescence and microscopy techniques.A labeled affinity reagent can be employed on a biological orhistological sample obtained from a subject. The affinity reagent ispreferably applied by overlaying the labeled affinity reagent onto thebiological sample and allowing the affinity reagent to contact anytarget BlaC protein that may be present. The sample is visualized underthe appropriate microscopy conditions to visualize the affinity reagentthrough its detectable label. Through this technique, it is possible todetermine not only the presence of the BlaC protein, but also itsdistribution within the sample. A wide variety of well-knownhistological methods can be utilized in order to achieve such in situdetection.

In another exemplary non-competitive immunoassay, the biological samplecan be brought in contact with, and immobilized onto, a solid phasesupport or a carrier, such as nitrocellulose, a plastic well, beads,magnetic particles, and the like. The solid phase support or carrier iscapable of immobilizing cells, cell particles or soluble proteins. Thesolid phase support or carrier can then be washed with suitable buffersfollowed by treatment with the detectably labeled affinity reagent. Thesolid phase support or carrier can then be washed with the buffer asecond time to remove unbound affinity reagent. The amount of boundlabel on solid phase support or carrier can then be detected byconventional means and is directly proportional to the amount of thetarget analyte, such as BlaC protein.

The term “solid phase support or carrier” is intended to mean anysupport or carrier capable of binding a cell or protein such as BlaC, oran affinity reagent. Well-known supports or carriers include glass,polystyrene, polypropylene, polyethylene, dextran, nylon, amylases,natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. A substrate that acts as a carrier can be either soluble tosome extent or insoluble for the purposes of the present invention. Thesupport or carrier material can have virtually any possible structuralconfiguration to conform to any assay format so long as the coupledtarget or affinity reagent is capable of binding to the correspondingaffinity reagent or target molecule, respectively. Thus, the support orcarrier configuration can be substantially spherical, as in a bead ormagnetic particle, or cylindrical, as in the inside surface of a testtube, or well in a multi-well plate. Alternatively, the surface can beflat such as a sheet, test strip, etc., that would be appropriate in alateral flow assay format. Those skilled in the art will recognize thatmany other suitable carriers are available for binding affinity reagentsor the target BlaC protein (or cells displaying the BlaC protein), orwill be able to ascertain the same by use of routine experimentation.

In some embodiments, the target protein/cell or affinity reagent isimmobilized directly to the solid phase support or carrier according tostandard protocols in the art. In other embodiments, the targetprotein/cell or affinity reagent is indirectly immobilized on the solidphase support or carrier. For example, as described in more detailbelow, antibodies can be “captured” and immobilized by protein A/G thatis bound to the solid support. Sometime it is preferable to utilizeknown blocking reagents to prevent spurious or elevated backgroundbinding.

For example, an illustrative format for the detection of BlaC in sputumis provided in the Snap Valve™ (Medical Packaging Corporation, CA, USA)that incorporates a flocked swab in a lateral flow device. The devicecan contain a matrix that can allow migration of the biological sample,including the target BlaC, past a region with immobilized affinityreagent. Detection of binding can be visualized as a result of any ofthe assay formats described herein, such as sandwich assays, competitiveassays, and the like.

In some embodiments, the target protein/cell or affinity reagent isconjugated onto a particle, such as a bead or magnetic particle, tofacilitate collection or immobilization for further analysis.

Another exemplary non-competitive immunoassay format is referred to as a“sandwich” assay. In a sandwich assay, one affinity reagent is typicallyimmobilized on a solid support or carrier. The biological sample iscaptured by the immobilized affinity reagent (thus, also referred to asthe “capture reagent”). A second affinity reagent (also referred to asthe “detection reagent”) that is detectably labeled is also added. Thecapture affinity reagent can be the same as the detection affinityreagent. For example, as described below, the same polyclonal antibodypopulation can be used for both the immobilization/capture and for thelabeled detection of the target BlaC protein. In other embodiments, thecapture affinity reagent can be different from the detection affinityreagent.

As used herein, the term “labeled” can refer to direct labeling of theaffinity reagent or target BlaC protein via, e.g., coupling a detectablesubstance to the affinity reagent or target protein. The term can alsorefer to indirect labeling of the affinity reagent by reactivity withanother affinity reagent that is directly labeled. For example, anantibody affinity reagent specific for BlaC protein can itself bespecifically bound by a second antibody that is detectably labeled.

In some embodiments, the detectable label comprises the coupling of anenzyme that is capable of producing a detectable signal when it actsupon a specific substrate. Some embodiments of enzyme-based immunoassaysare referred to as enzyme linked immunosorbent assays (ELISAs) and arewell-known in the art. See e.g., Voller, A., “The Enzyme LinkedImmunosorbent Assay (ELISA),” 1978, Diagnostic Horizons 2:1-7,Microbiological Associates Quarterly Publication, Walkersville, Md.;Voller, A. et al., 1978, J. Clin. Pathol. 31:507-520; Butler, J. E.,1981, Meth. Enzymol. 73:482-523; Maggio, E. (ed.), 1980, EnzymeImmunoassay, CRC Press, Boca Raton, Fla.; Ishikawa, E. et al., (eds.),1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo). The enzyme which is boundto the antibody will react with an appropriate substrate, preferably achromogenic substrate, in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorimetricor by visual means. Enzymes which can be used to detectably label theantibody include, but are not limited to, malate dehydrogenase,staphylococcal nuclease, delta-5-steroid isomerase, yeast alcoholdehydrogenase, alpha-glycerophosphate, dehydrogenase, triose phosphateisomerase, horseradish peroxidase, alkaline phosphatase, asparaginase,glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The substrates for these illustrative enzymes arecommonly known in the art. The detection can be accomplished bycolorimetric methods which employ a chromogenic substrate for theenzyme. Detection can also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

An exemplary protocol for a sandwich ELISA format in a multi-well plateis as follows: 1) coat plate with anti-BlaC capture antibody; 2) block(and optionally preserve and dry) the coated plate; 3) add biologicalsample in buffer to the plate and incubate for approximately 30 minutes;4) wash; 5) add anti-BlaC-HRP affinity reagent to plate; 6) wash; 7) addHRP substrate to plate and incubate for approximately 15 minutes; and 8)add stop solution and read signal to determine amount of bound targetBlaC on the plate. It will be readily recognized that variousalterations to the above protocol can be made. One variation in theassay format includes pre-incubating the detection affinity reagent andbiological sample before adding to the plate. Other variations are knownand commercially available. For instance, one illustrative assay formatis the Simoa™ assay (Quanterix, MA, USA), which incorporates the ELISAapproach on a nanoscale using affinity reagents attached to paramagneticparticles. The particles are then loaded individually intofemtoliter-scale wells and read for signal.

In other embodiments, the target protein or affinity reagent can bedirectly coupled to detectable moieties. For example, in aradioimmunoassay (RIA) the target protein or affinity reagent can beradioactively labeled, allowing detection of the target protein throughany of the described formats. The radioactive isotope (e.g., 1251, 1311,35S or 3H) can be detected by such means as the use of a gamma counteror a scintillation counter or by autoradiography.

In other embodiments, the target BlaC protein or affinity reagent iscoupled to a fluorescent compound. When the fluorescently labeledantibody is exposed to light of the proper wavelength, its presence canthen be detected due to fluorescence. A non-limiting, illustrative listof fluorescent compounds includes fluorescein isothiocyanate, rhodamine,phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde andfluorescamine.

In other embodiments, the target BlaC protein or affinity reagent iscoupled to a fluorescence emitting metal such as 152Eu, or others of thelanthanide series. These metals can be attached to the antibody usingsuch metal chelating groups as diethylenetriaminepentacetic acid (DTPA)or ethylenediaminetetraacetic acid (EDTA).

In other embodiments, the target BlaC protein or affinity reagent isconjugated to a chemiluminescent compound. The presence of thechemiluminescent-tagged target BlaC protein or affinity reagent is thendetermined by detecting the presence of luminescence that arises duringthe course of a chemical reaction. Illustrative, non-limiting examplesof particularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

In other embodiments, the target BlaC protein or affinity reagent isconjugated to a bioluminescent compound. Bioluminescence is a type ofchemiluminescence found in biological systems in which a catalyticprotein increases the efficiency of the chemiluminescent reaction. Thepresence of a bioluminescent protein is determined by detecting thepresence of luminescence. Important bioluminescent compounds forpurposes of labeling are luciferin, luciferase and aequorin. The proteincan also be detected by monitoring its catalytic activity (such as inELISA), if the protein is an enzyme. The protein can also be detectedusing coupled enzymatic assays.

In other embodiments, the affinity reagent contains a fluorescentprotein domain. Such fluorescent protein domains are known in the art,and include GFP, dtTomato, and mCherry. The affinity reagent with afluorescent protein domain can be produced according to any commonlyknown techniques, such as the recombinant expression of a fusion proteinthat includes the fluorescent domain.

According to this aspect of the disclosure, the presence of BlaC in thebiological sample, as determined according to any recognized methodincluding those described above, is indicative of a TB-complex bacteriumin the sample. As used herein, the term “sample” refers to any samplethat can harbor bacteria or bacterial secretions, and can includefluids, pharmaceutical compositions, or consumable products, which couldpotentially be contaminated with mycobacteria. In some embodiments, thesample is a biological sample, such as comprising nutrients and/or mediasufficient to support sustenance or growth of living mycobacteria. Insome embodiments, the biological sample is a tissue culture sample.

In a further aspect of the disclosure, the sample is a biological sampleobtained from a subject. According to this aspect, the presence of BlaCin the biological sample obtained from a subject, as determinedaccording to any recognized method including those described above, isindicative of a tuberculosis infection in the subject.

As used herein, the term “subject” refers to any animal that can harbora tuberculosis infection by any of the described bacteria in theTB-complex. In some embodiments, the subject is a vertebrate animal. Insome embodiments, the subject is a mammal. In some embodiments thesubject is a human.

Suitable biological samples include sputum, pleural fluid, spinal fluid,blood, urine, saliva, stool/feces, mucus, tissue biopsies, tissuehomogenates, directly in live animals or human patients, or a sampleobtained by swabbing an area of interest on a subject. For example,illustrative examples of BlaC and/or bacteria expressing BlaC detectionusing the disclosed reagents were performed in sputum and salivasamples, as described in more detail below.

In another aspect, the disclosure provides a method for detecting thepresence or amount of anti-β-lactamase (BlaC) antibody in a biologicalsample obtained from a subject. A determined presence ofanti-β-lactamase (BlaC) antibody in the sample is indicative of thepresence of BlaC in the subject, and hence, is indicative of aTB-complex bacterial infection in the subject. In some embodiments, suchan assay can incorporate the use of the antigen/epitope unique to BlaCover somewhat similar β-lactamase antigens. For example, aswell-understood in the art, such BlaC antigen/epitopes can beimmobilized to a solid substrate whereby the antigen/epitopes arecontacted with the biological sample. Binding of any antibody from thesample can be detected, for example, using a labeled antibody specificfor antibodies from the subject.

It will be recognized that the subject's antibody can form a complexwith antigenic fragments of the BlaC protein. Antigenic portions willtypically comprise at least six amino acids of the BlaC protein.Specifically, the antigenic portions typically comprise the amino acidsthat are exposed to the exterior environment of the expressed protein,such that they are accessible to the B-cells of the subject's immunesystem. Accordingly, in some embodiments, the method comprises (a)contacting the biological sample with at least one polypeptide with anamino acid sequence that has at least 90% sequence identity to any sixor more amino acids of SEQ ID NO:2; and (b) detecting the formation of acomplex between the antibody in the sample and the polypeptide. In someembodiments, the at least one polypeptide has an amino acid sequencewith at least 90′% sequence identity to at least six to 20, or moreamino acids of SEQ ID NO:2. In further embodiments, the amino acids arecontiguous. It will also be appreciated that the polypeptide willpreferably have a unique sequence, or a low sequence identity to otherbacterial β-lactamases, so as to avoid forming a complex under thestandard conditions. In some embodiments, the polypeptide, or fragmentthereof, comprises amino acids corresponding to amino acids withinpositions 75 to 87 of SEQ ID NO:2, within amino acids at positions 142to 144 of SEQ ID NO:2, and/or within amino acids at positions 249 to 252of SEQ ID NO:2. Such regions are known to participate in structuralmotifs that are important for the unique enzymatic activity of BlaC (seeFIG. 5).

Detection of a complex between the antibody and the polypeptide can beaccomplished with any method known in the art for this purpose,including those described herein above. For example, antibodies in thebiological sample can be immobilized on a substrate and BlaC (or BlaCfragment) with a detectable label attached thereto can be added.Conversely, the BlaC (or BlaC fragment) can be immobilized on thesubstrate and the biological sample contacted thereto. A detectionaffinity reagent specific for human antibodies can be applied after awash cycle. The retention of a detectable signal after a wash cycle isindicative that the subject has produced an anti-β-lactamase antibody.

In some embodiments, the method further comprises comparing a determinedamount of anti-BlaC antibody (by virtue of detectable signal intensity)to a reference standard to establish the level of binding with respectto background signal. An amount of anti-BlaC antibody detected in thebiological sample greater than the reference standard is indicative ofthe presence or relative amount of tuberculosis-complex bacteria in thesubject. It is preferred that the reference standard is a biologicalsample-type that is the same as the biological sample obtained from thesubject.

In another aspect, the present disclosure provides an assay kitcomprising the affinity reagent described herein. In some embodiments,the assay kit also includes buffers and requisite reagents as describedherein to analyze a biological sample for the presence of BlaC. In someembodiments, the kit includes a device that provides a solid support. Insome embodiments, the kit can comprise a lateral flow device. In someembodiments, the kit can comprise an ELISA format plate.

As described herein, the blaC gene encoding the β-lactamase in theTB-complex bacteria of M. tuberculosis has unique domains that aredissimilar to the homologous bla genes encoding β-lactamase in otherpathogenic bacteria, including non TB-complex bacteria in the genusMycobacteria. Accordingly, in another aspect, the present disclosureprovides a method for presence of TB-complex bacteria in a test sampleby virtue of the detection of the unique, TB-complex specificβ-lactamase sequence at the nucleic acid level.

In one embodiment, the method comprises contacting the sample with apolynucleotide probe capable of specifically hybridizing to a targetregion of a nucleic acid molecule that encodes a β-lactamase (BlaC) withan amino acid sequence set forth in SEQ ID NO:2. The method furthercomprises detecting the hybridization of the probe to the nucleic acidmolecule encoding BlaC, wherein detected hybridization is indicative ofthe presence of tuberculosis complex bacteria in the test sample. Insome embodiments, the probe comprises a detectable label.

As used herein, the terms “nucleic acid, polynucleic acid, orpolynucleotide” refer to a deoxyribonucleotide polymer (i.e., DNA) orribonucleotide polymer (i.e., RNA) in either single- or double-strandedform. Unless otherwise limited, encompasses known analogs of naturalnucleotides that hybridize to nucleic acids in a manner similar tonaturally occurring nucleotides, such as peptide nucleic acids (PNAs)and phosphorothioate DNA. Unless otherwise indicated, a particularnucleic acid sequence includes the complementary sequence thereof.Nucleotides include, but are not limited to, ATP, dATP, CTP, dCTP, GTP,dGTP, UTP, TTP, dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, dITP,2-amino-adenosine-TP, 2-amino-deoxyadenosine-TP, 2-thiothymidinetriphosphate, pyrrolo-pyrimidine triphosphate, and 2-thiocytidine, aswell as the alphathiotriphosphates for all of the above, and2′-O-methyl-ribonucleotide triphosphates for all the above bases.Modified bases include, but are not limited to, 5-Br-UTP, 5-Br-dUTP,5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and 5-propynyl-dUTP.

As used herein, the term “polynucleotide probe” refers to a nucleic acidwith a plurality of nucleotide subunits in a contiguous polymer chain.The term “capable of specifically hybridizing” with respect to thepolynucleotide probe refers to the ability of the polynucleotide probe,by virtue of its nucleotide sequence, to form and maintain non-covalentbonds between the nucleotides of opposing polymer strands. For example,in DNA the pyrimidines base structures thymine (T) and cytosine (C)typically pair with the purine base structures adenine (A) and guanine(G), respectively. It is recognized that some mismatch in sequencebetween the probe and the template polynucleic probe is permitted.However, the sequences must be sufficiently complementary to permithybridization (“annealing”) under standard conditions. Standardhybridization conditions are known in the art. Thus, the term“specifically hybridize” as used herein refers to the ability of anucleic acid to hybridize detectably and specifically to a secondnucleic acid. Polynucleotides specifically hybridize with target nucleicacid strands under standard hybridization and wash conditions thatminimize appreciable amounts of detectable binding to non-specificnucleic acids (i.e., without corresponding sequence similarity).

In this aspect, the probe specifically hybridizes to a target region ofa nucleic acid molecule that encodes a β-lactamase (BlaC) with an aminoacid sequence set forth in SEQ ID NO:2. Skilled artisans knowing theredundancy of the genetic code will understand the scope of nucleicacids (e.g., DNA and RNA molecules) that can encode the amino acidsequence in SEQ ID NO:2. In one embodiment, the nucleic acid moleculethat encodes a β-lactamase (BlaC) is set forth in SEQ ID NO: 1.

In some embodiments, the target region is at least 10 contiguousnucleotides of the encoding nucleic acid.

It will be appreciated that the sequence of the target region will havelow sequence identity with any nucleic acid sequence appearing innon-TB-complex bacteria. As used herein with respect to nucleic acidmolecules, the term “sequence identity” or “percent identical” is thepercentage of nucleic acid residues in a candidate nucleic acid moleculesequence that are identical with a subject nucleic acid moleculesequence (such as the nucleic acid molecule sequence set forth in SEQ IDNO:2), after aligning the sequences to achieve the maximum percentidentity, and not considering any nucleic acid residue substitutions aspart of the sequence identity. No gaps are introduced into the candidatenucleic acid sequence in order to achieve the best alignment. Nucleicacid sequence identity can be determined in the following manner. Thesubject polynucleotide molecule sequence is used to search a nucleicacid sequence database, such as the Genbank database, using the programBLASTN version 2.1 (based on Altschul et al., Nucleic Acids Research25:3389-3402 (1997)). The program is used in the ungapped mode. Defaultfiltering is used to remove sequence homologies due to regions of lowcomplexity as defined in Wootton, J. C., and S. Federhen, Methods inEnzymology 266:554-571 (1996). The default parameters of BLASTN areutilized. It will be appreciated that “low sequence identity” willresult in a failure of the probe to hybridize under standard conditionswith the non-target region of the nucleic acid from non-TB-complexsources.

In some embodiments, the polynucleotide probe comprises a polynucleotidesequence selected from the group consisting of SEQ ID NO:3, 4, 5, 6, 7,8, 9, 10, and 11. In some embodiments, the polynucleotide probeencompasses homologs of the polynucleotides selected from the groupconsisting of SEQ ID NO:3, 4, 5, 6, 7, 8, 9, 10, and 11. As used herein,the term “homologs” refers to nucleic acids having one or morealterations in the primary sequence set forth in any one of SEQ ID NO:3,4, 5, 6, 7, 8, 9, 10, and 11, that does not destroy the ability of thepolynucleotide to specifically hybridize with a target sequence, asdescribed above. A primary sequence can be altered, for example, by theinsertion, addition, deletion or substitution of one or more of thenucleotides. Thus, in some embodiments, the polynucleotide probecomprises a polynucleotide sequence that is at least about 90%, 95%, or99% identical to a sequence selected from the group consisting of SEQ IDNO:3, 4, 5, 6, 7, 8, 9, 10, and 11.

Detection of hybridization of the probe to the nucleic acid moleculeencoding BlaC can be accomplished through any commonly known technique.For example, the probe may be configured to emit a detectable signalonly upon specific binding to the nucleic acid target sequence. In oneillustrative probe configuration, known as a molecular beacon probe, theprobe maintains a hairpin/loop shape. The internal loop domain comprisesthe sequence that is complementary to the target nucleic acid sequence.The stem of the hairpin structure is formed by complementaryoligonucleotide sequences that are at the 5′ and 3′ end of the linearprobe sequence. A fluorophore is covalently attached to the end of oneof the stem oligonucleotide sequences, whereas a quencher dye iscovalently attached to the end of the other stem oligonucleotide. Whenin the intact hairpin configuration, the probe does not emit adetectable signal because of the close proximity of the quencher andfluorophore. However, when annealing to the target sequence, the probelinearizes permitting a sufficient distance to form between thefluorophore and quencher dyes, thus allowing a detectable signal. Such aprobe can be incorporated into extension or amplification-based assays,such as PCR-based amplification assays, as described in more detailbelow.

In another embodiment of this aspect, the disclosure provides a methodfor determining the presence of tuberculosis-complex bacteria in a testsample, which generally comprises the following steps: (a) contactingthe test sample with a composition comprising at least one primer paircomprising a forward and reverse primer capable of specificallyhybridizing to a target region of tuberculosis-complex blaC gene, toform a reaction mixture; (b) subjecting said reaction mixture toamplification conditions suitable to amplify at least a portion of thetarget region; and (c) detecting amplification of the at least a portionof the target region, wherein amplification of the at least a portion ofthe target region is indicative of the presence of tuberculosis-complexbacteria in a test sample.

As used herein, the term “primer” means a polynucleotide which can serveto initiate a nucleic acid chain extension reaction. Typically, primershave a length of 5 to about 50 nucleotides, although primers can belonger than 50 nucleotides. Accordingly, in some embodiments, additionalconditions and reagents are provided to facilitate the extensionreaction using the probe as a primer. In some embodiments, the probe isone of a primer pair, such as used in the polymerase chain reaction.When employed as part of a primer pair, the probe can facilitatesuccessive rounds of extension of the nucleic acid molecule sequence.Each successive round produced an increase of template nucleic acidtemplate, thus leading to the amplification of the sequence. In suchembodiments, a detectable label can include moieties or chemicals thatare not linked to the probe. For example, dyes such as ethidium bromideor SYBR green intercalate in double stranded DNA and can serve as anindicator of successful amplification.

It will be appreciated that certain selection criteria are preferablyemployed when selecting primers (and optional probes). For example, forprimer pairs for use in the amplification reactions, the primers areselected such that the likelihood of forming 3′ duplexes is minimized,and such that the melting temperatures (Tm) are sufficiently similar tooptimize annealing to the target sequence and minimize the amount ofnon-specific annealing. In this context, the polynucleotides accordingto the present invention are provided in combinations that can be usedas primers in amplification reactions to specifically amplify targetnucleic acid sequences. Furthermore, it will be appreciated that tospecifically hybridize with the target region of the nucleic acidmolecule, the sequence identity with other non-TB-complex bla genesequences will be low.

In some embodiments, the forward primer has a polynucleotide sequenceselected from the group consisting of SEQ ID NO:12, 13, 14, 15, 16, 17,18, 19, 20, and any homologue thereof. Thus, in some embodiments, thepolynucleotide probe comprises a polynucleotide sequence that is atleast 90% identical to a sequence selected from the group consisting ofSEQ ID NO:12, 13, 14, 15, 16, 17, 18, 19, and 20.

In some embodiments, the reverse primer has a polynucleotide sequenceselected from the group consisting of SEQ ID NO:21, 22, 23, 24, 25, 26,27, 28, 29, and any homologue thereof. Thus, in some embodiments, thepolynucleotide probe comprises a polynucleotide sequence that is atleast 90% identical to a sequence selected from the group consisting ofSEQ ID NO:21, 22, 23, 24, 25, 26, 27, 28, and 29.

In some embodiments, the primer pair has sequences selected from thegroup consisting of SEQ ID NOS:12 and 21 (or homologs thereof), SEQ IDNOS:13 and 22 (or homologs thereof), SEQ ID NOS:14 and 23 (or homologsthereof), SEQ ID NOS:15 and 24 (or homologs thereof), SEQ ID NOS:16 and25 (or homologs thereof), SEQ ID NOS:17 and 26 (or homologs thereof),SEQ ID NOS:18 and 27 (or homologs thereof), SEQ ID NOS:19 and 28 (orhomologs thereof), and SEQ ID NOS:20 and 29 (or homologs thereof).

Extension and amplification procedures are well-known in the art andinclude, but are not limited to, polymerase chain reaction (PCR), TMA,rolling circle amplification, nucleic acid sequence based amplification(NASBA), and strand displacement amplification (SDA). One skilled in theart will understand that for use in certain amplification techniques theprimers may need to be modified, for example, for SDA the primercomprises additional nucleotides near its 5′ end that constitute arecognition site for a restriction endonuclease. Similarly, for NASBAthe primer comprises additional nucleotides near the 5′ end thatconstitute an RNA polymerase promoter. Polynucleotides thus modified areconsidered to be within the scope of the present invention.

Nucleic acid amplification reagents include reagents, which are wellknown and may include, but are not limited to, an enzyme having at leastpolymerase activity, enzyme cofactors such as magnesium or manganese;salts; nicotinamide adenine dinucleotide (NAD); and deoxynucleotidetriphosphates (dNTPs) such as for example deoxyadenine triphosphate,deoxyguanine triphosphate, deoxycytosine triphosphate and deoxythyminetriphosphate.

Amplification conditions are conditions that generally promote annealingand extension of one or more target nucleic acid sequences. It is wellknown that such annealing is dependent in a rather predictable manner onseveral parameters, including temperature, ionic strength, sequencelength, complementarity, and G:C content of the sequences. For example,lowering the temperature in the environment of complementary nucleicacid sequences promotes annealing. For any given set of sequences, melttemperature, or Tm, can be estimated by any of several known methods.Typically, diagnostic applications utilize hybridization temperaturesthat are about 10° C. (e.g., 2° C. to 18° C.) below the melttemperature. Ionic strength or “salt” concentration also impacts themelt temperature, since small cations tend to stabilize the formation ofduplexes by negating the negative charge on the phosphodiester backbone.Typical salt concentrations depend on the nature and valency of thecation but are readily understood by those skilled in the art.Similarly, high G:C content and increased sequence length are also knownto stabilize duplex formation because G:C pairings involve 3 hydrogenbonds where A:T pairs have just two, and because longer sequences havemore hydrogen bonds holding the sequences together. Thus, a high G:Ccontent and longer sequence lengths impact the hybridization conditionsby elevating the melt temperature.

Specific amplicons produced by amplification of target nucleic acidsequences using the polynucleotides of the present invention, asdescribed above, can be detected by a variety of methods known in theart. For example, one or more of the primers used in the amplificationreactions may be labeled such that an amplicon can be directly detectedby conventional techniques during or subsequent to the amplificationreaction. In another embodiment, total amplified product can beascertained by inclusion of specific dyes, such as SYBR green, or anantibody that specifically detects the amplified nucleic acid sequence.In yet another embodiment, a third polynucleotide distinct from theprimer sequences that has been labeled and is complementary to a regionof the amplified sequence, can be added during or after theamplification reaction is complete. This third polynucleotide can be theprobe as described above.

As indicated, the amplification products produced as described above canbe detected during or subsequently to the amplification of the targetsequence. Methods for detecting the amplification of a target sequenceduring amplification are outlined above, and described, for example, inU.S. Pat. No. 5,210,015. Gel electrophoresis can be employed to detectthe products of an amplification reaction after its completion.Alternatively, amplification products are hybridized to probes, thenseparated from other reaction components and detected usingmicroparticles and labeled probes. However, it will be readilyappreciated both amplification and detection of target nucleic acidsequences can also take place concurrently in a single unopened reactionvessel. This type of procedure allows “real-time” monitoring of theamplification reaction, “end-point” monitoring, and can avoidcontamination by reducing the handling steps.

For embodiments in which both amplification with polynucleotide primersand distinct detection probes are included concurrently during theamplification reaction, the polynucleotide probe preferably possessescertain properties. For example, since the probe will be present duringthe amplification reaction, it should not interfere with the progress ofthis reaction and should also be stable under the reaction conditions.In addition, for real-time monitoring of reactions, the probe should becapable of binding its target sequence under the conditions of theamplification reaction and to emit a signal only upon binding thistarget sequence. Examples of probe molecules that are particularlywell-suited to this type of procedure include molecular beacon probesand probes comprising a fluorophore covalently attached to the 5′ end ofthe probe and a quencher at the 3′ end (e.g., TaqMan® probes).

The present invention, therefore, contemplates the use of thepolynucleotides as TaqMan® probes as demonstrated below and illustratedin FIG. 12. As is known in the art, TaqMan® probes are dual-labeledfluorogenic nucleic acid probes composed of a polynucleotidecomplementary to the target sequence that is labeled at the 5′ terminuswith a fluorophore and at the 3′ terminus with a quencher. TaqMan®probes are typically used as real-time probes in amplificationreactions. In the free probe, the close proximity of the fluorophore andthe quencher ensures that the fluorophore is internally quenched. Duringthe extension phase of the amplification reaction, the probe is cleavedby the 5′ nuclease activity of the polymerase and the fluorophore isreleased. The released fluorophore can then fluoresce and thus producesa detectable signal.

The term “detectable label” as used herein with reference to polynucleicacids refers to a molecule or moiety having a property or characteristicwhich is capable of detection and, optionally, of quantitation. Similarto labels described above in the context of detectably labeled proteins,a label can be directly detectable, as with, for example (and withoutlimitation), radioisotopes, fluorophores, chemiluminophores, enzymes,colloidal particles, fluorescent microparticles and the like; or a labelmay be indirectly detectable, as with, for example, specific bindingmembers. It will be understood that directly detectable labels mayrequire additional components such as, for example, substrates,triggering reagents, quenching moieties, light, and the like to enabledetection and/or quantitation of the label. When indirectly detectablelabels are used, they are typically used in combination with a“conjugate.” A conjugate is typically a specific binding member that hasbeen attached or coupled to a directly detectable label. Methods oflabeling nucleic acid sequences are well known in the art (see, forexample, Ausubel et al. (1997 & updates), Current Protocols in MolecularBiology, Wiley & Sons, New York). For example, coupling chemistries forsynthesizing a conjugate are well known in the art and can include, forexample, any chemical means and/or physical means that does not destroythe specific binding property of the specific binding member or thedetectable property of the label.

Suitable fluorophores quenchers for use with various embodiments ofpolynucleotides of the present invention can be readily determined byone skilled in the art (see also Tyagi et al., Nature Biotechnol.,16:49-53 (1998); Marras et al., Genet. Anal. Biomolec. Eng., 14:151-156(1999)). Many fluorophores and quenchers are available commercially, forexample from Molecular Probes (Eugene, Oreg.) or Biosearch Technologies,Inc. (Novato, Calif.). Examples of fluorophores that can be used in thepresent invention include, but are not limited to, fluorescein andfluorescein derivatives such as carboxy fluorescein (FAM®), a dihalo-(C1to C8)dialkoxycarboxyfluorescein,5-(2′-aminoethyl)aminonaphthalene-1-sulphonic acid (EDANS), coumarin andcoumarin derivatives, Lucifer yellow, Texas red, tetramethylrhodamine,tetrachloro-6-carboxyfluoroscein, 5-carboxyrhodamine, cyanine dyes andthe like. Quenchers include, but are not limited to, DABCYL,4′-(4-dimethylaminophenylazo)benzoic acid (DABSYL),4-dimethylaminophenylazophenyl-4-dimethylaminophenylazophenyl-4′-maleimide(DABMI), tetramethylrhodamine, carboxytetramethylrhodamine (TAMRA),dihydrocyclopyrroloindole tripeptide minor groover binder (MGB®) dyesand the like.

In some embodiments that combine the use of primers and probes, theprimer pair/probe combination has sequences selected from the groupconsisting of SEQ ID NOS:3, 12, and 21 (or homologs thereof), SEQ IDNOS:4, 13, and 22 (or homologs thereof), SEQ ID NOS:5, 14, and 23 (orhomologs thereof), SEQ ID NOS:6, 15, and 24 (or homologs thereof), SEQID NOS:7, 16, and 25 (or homologs thereof), SEQ ID NOS:8, 17, and 26 (orhomologs thereof), SEQ ID NOS:9, 18, and 27 (or homologs thereof), SEQID NOS:10, 19, and 28 (or homologs thereof), and SEQ ID NOS:11, 20, and29 (or homologs thereof). Such combinations of primer pair/probes areset forth below in Tables 1 and 2.

Any polynucleotide according to the present invention can be prepared byconventional techniques well known to those skilled in the art. Forexample, the polynucleotides can be prepared using conventionalsolid-phase synthesis using commercially available equipment, such asthat available from Applied Biosystems USA Inc. (Foster City, Calif.),DuPont (Wilmington, Del.), or Milligen (Bedford, Mass.). Modifiedpolynucleotides, such as phosphorothioates and alkylated derivatives,can also be readily prepared by similar methods known in the art. See,for example, U.S. Pat. Nos. 5,464,746; 5,424,414; and 4,948,882.

In some embodiments, the test sample is obtained from a subject, asdescribed above. In these embodiments, the presence of TB-complexbacteria in the test sample is indicative of a TB-complex bacterium inthe subject. The subject can be a human or animal suspected of having alatent or active TB infection. Alternatively, the subject can be alaboratory model for infection with TB and TB-complex bacteria. Thus,the method is useful for studying the progression and transmission ofTB-complex bacteria.

In other embodiments, the test sample is from a culture, such as tissueor cell culture. The disclosed method is useful for establishingcontamination, or for monitoring the in vitro culturing of theTB-complex bacteria.

In another aspect, the present disclosure provides a method formonitoring the efficacy of treatment of a tuberculosis infection. Themethod comprises (a) determining the presence or amount of BlaC protein,nucleic acid encoding BlaC protein, or anti-BlaC antibodies in abiological sample obtained from a subject receiving treatment fortuberculosis according to the above descriptions; and (b) comparing theamount of BlaC protein, nucleic acid encoding BlaC protein, or anti-BlaCantibodies in the biological sample as determined in step (a) to areference standard.

In some embodiments, the reference standard in step (b) is the amount ofBlaC protein, nucleic acid encoding BlaC protein, or anti-BlaCantibodies determined in an analogous biological sample obtained fromthe subject at or after diagnosis with the tuberculosis infection butprior to the obtaining of the biological sample from the subject in step(a). A lower amount of anti-BlaC antibodies in the biological sampledetermined in step (a) compared to the biological sample in step (b) isindicative of a positive response to the treatment. In some embodiments,the reference standard is determined from a biological sample obtainedfrom the subject at or prior to the commencement of treatment for thetuberculosis infection.

It will be appreciated that the applicable subjects and biologicalsamples described above are equally applicable to the present aspects ofthe invention directed to detection of blaC sequence.

In another aspect, the present disclosure provides an isolatedpolynucleic acid with a detectable label covalently coupled thereto,wherein the isolated polynucleic acid is capable of hybridizing to atarget region of a blaC gene encoding the amino acid sequence set forthin SEQ ID NO:2. In some embodiments, the isolated polynucleic acidcomprises a nucleic acid sequence set forth in any one of SEQ IDNOS:3-29, or a homolog or variant thereof with about at least 90%, 95%,96%, 97%, 98%, or 99% sequence identity thereto.

In some embodiments, the isolated polynucleotide further comprises aquencher moiety covalently coupled thereto.

In another aspect, the present disclosure provides a kit that comprisesat least one of the isolated polynucleic acid molecules describedimmediately above. In some embodiments, the kit further comprisesadditional reagents to facilitate hybridization of the isolatedpolynucleic acid molecules to the target region of a blaC gene encodingthe amino acid sequence set forth in SEQ ID NO:2, as described herein.In some embodiments, the kit includes primer oligonucleotides, asdescribed herein, that are capable of amplifying a portion of the blaCgene encoding the amino acid sequence set forth in SEQ ID NO:2 under theconditions described herein.

In conclusion, the compositions, methods and systems described hereinare useful to detect and monitor TB-bacteria. Many illustrativeembodiments have been described, but the disclosure is not so limited.It will be appreciated by persons of skill in the art that many of thecompounds, reagents, methods, systems, and kits described andcontemplated herein can be incorporated with a variety of commonlyrecognized assay formats and their integral components, such asmicrofluidic systems, mass spectrometry systems, nanoparticle systems,microscopy systems, and the like. For example, microfluidics systems canbe used to trap the protein and facilitate detection with antibodies orprobes, mass spectroscopy, or other detection methods. Nanoparticle ornanopore systems can be developed using mirror thin films or particlesthat can be made to specifically detect proteins with structural andelectrostatic properties similar to BlaC followed by mass spectroscopy,colorimetric, electronic or antibody-based detection of the protein.Microscopy could be used in combination with antibodies against BlaC todetect individual or clumps of bacteria augmenting smear microscopyalready used and enhancing detection or improving specificity of currenttests. These approaches can be used in combination with fluorescent orcolorimetric antibody methods to detect the protein or intact bacteriain samples on slides. Magnetic bead separation can be used to allow moresensitive detection of BlaC in nearly any clinical material. Detectioncan be performed using FACS, microscopy, plate reader, MS, and the like.

Similarly, the described nucleic acid-based methods reagents can bedeveloped and incorporated into a variety of known DNA or RNA analysissystems, such as qRT-PCR assays, molecular beacon assays, solid supportsystems, nanoparticles or thin films that carry at least a portion ofthe specific primers. Other strategies can be incorporated into a testsystem, including RNA primers, antibodies directed against nucleotidecomplexes, and hybridization complexes that produce colorimetric,fluorescent or luminescent output. Microfluidics systems can be used totrap the specific DNA or RNA for blaC and detection could be throughPCR-like systems, hybridization to indicator probes or other automatedstrategies, including imaging techniques, FISH, scanning-tunnelingmicroscopy, and electronic detection of hybridization. Magnetic beadseparation could be used to improve yields of the target and increasethe sensitivity of PCR, qRT-PCR or other detection methods. Detection ofthe blaC sequence can be accomplished using FACS, microscopy, platereader, MS, and the like. An RNA-based test could have the advantagethat it would allow measurement of viability due to the half-life of theblaC RNA transcript, which could be applied to evaluate therapeuticoutcome and as the basis of a drug-susceptibility test (DST).

Unless specifically defined herein, all terms used herein have the samemeaning as they would to one skilled in the art of the presentinvention. Practitioners are particularly directed to Sambrook et al.,(1989) Molecular Cloning: A Laboratory Manual, 2d ed., Cold SpringHarbor Press, Plainsview, N.Y.; and Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York (1999) for definitionsand terms of art.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Following long-standing patent law, the words “a” and “an,” when used inconjunction with the word “comprising” in the claims or specification,denotes one or more, unless specifically noted.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise,” “comprising,” and thelike, are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to indicate, in the sense of“including, but not limited to.” Words using the singular or pluralnumber also include the plural and singular number, respectively.Additionally, the words “herein,” “above,” and “below,” and words ofsimilar import, when used in this application, shall refer to thisapplication as a whole and not to any particular portions of theapplication.

Disclosed are materials, compositions, and components that can be usedfor, can be used in conjunction with, can be used in preparation for, orare products of the disclosed methods and compositions. It is understoodthat, when combinations, subsets, interactions, groups, etc., of thesematerials are disclosed, each of various individual and collectivecombinations is specifically contemplated, even though specificreference to each and every single combination and permutation of thesecompounds may not be explicitly disclosed. This concept applies to allaspects of this disclosure including, but not limited to, steps in thedescribed methods. Thus, specific elements of any foregoing embodimentscan be combined or substituted for elements in other embodiments. Forexample, if there are a variety of additional steps that can beperformed, it is understood that each of these additional steps can beperformed with any specific method steps or combination of method stepsof the disclosed methods, and that each such combination or subset ofcombinations is specifically contemplated and should be considereddisclosed. Additionally, it is understood that the embodiments describedherein can be implemented using any suitable material such as thosedescribed elsewhere herein or as known in the art.

Publications cited herein and the subject matter for which they arecited are hereby specifically incorporated by reference in theirentireties.

The following disclosures provide illustrations of various aspects ofthe present disclosure. These disclosures are illustrative only and areunderstood to not be limiting to the spirit and scope of the disclosure.

I. The following disclosure describes a study demonstrating the specificand sensitive detection of Mycobacterium tuberculosis β-lactamase (BlaC)and TB complex bacteria expressing β-lactamase protein in biologicalsamples using antibody-based immune-assay techniques.

β-Lactamase (BlaC) Protein Generation

Escherichia coli harboring an expression plasmid for Mycobacteriumtuberculosis β-lactamase (BlaC) was cloned from M. tuberculosis genomicDNA. The gene, as set forth Gene ID: 888742 of GenBank genome accessionno. NC_000962.3, was amplified using the polymerase chain reaction undernormal conditions (see description of the original cloning of the genein Wang, F. et al., “Crystal structure and activity studies of theMycobacterium tuberculosis β-lactamase reveal its critical role inresistance to β-lactam antibiotics,” Antimicrobial Agents andChemotherapy 50(8):2762-2771 (2006), incorporated herein by reference inits entirety). The amplified blaC gene was subsequently inserted intothe pET28b vector using NdeI and HindIII restriction sites. Theresultant plasmid was transformed into E. coli BL21 strain. Thetransformed E. coli cells were validated and used for expression andpurification of BlaC.

The E. coli strain was cultured in Luria-Bertani-Miller (LB) media (BDBiosciences, California, USA) containing 50 μg/ml of kanamycin, at 37°C. to obtain an optical density (OD₆₀₀) of 0.6. Expression of BlaC wasthen induced by the addition of isopropyl β-D-thiogalactopyranoside(IPTG, Gold Biotechnology, MO, USA) at 4° C. for 16 h. The cells werethen harvested by centrifugation at 10,000 g and 4° C. for 10 minutes.The pellets were re-suspended in 25 mM Tris-HCl (pH 8.0) and lysed usingthree repetitive cycles of freeze-thaw followed by the addition of 2.5unit/ml of benzonase (Novagen®, Darmstadt, Germany). The lysate wasloaded onto a His-Prep™ column (GE Health Care, Buckinghamshire, UK),purification of BlaC was carried out according to the previouslydescribed protocol (Wang, F. et al., “Crystal structure and activitystudies of the Mycobacterium tuberculosis β-lactamase reveal itscritical role in resistance to β-lactam antibiotics,” AntimicrobialAgents and Chemotherapy 50(8):2762-2771 (2006), incorporated herein byreference in its entirety), using the ÄKTA pure system (GE Health Care,Buckinghamshire, UK). Purified BlaC concentration of 4 mg/ml was finallyachieved by dialysis with Spectra/Por® dialysis tubes (Spectrum Labs,TX, USA).

To validate the expression of BlaC from E. coli and the quality of thepurified protein, samples were collected at various stages during thepurification and run on a 12% SDS-polyacrylamide gel. It was observedthat the amount of BlaC in the E. coli culture increased substantiallyfollowing IPTG induction. Further, it is illustrated that upon lysis ofthe cells, maximal protein was retained in solution and not in the celldebris.

Characterization of Recombinant β-Lactamase (BlaC)

As described, the purified BlaC protein was run on a 120% SDS-PAGE tocheck for purity and molecular weight. A single band was observedcorresponding to molecular weight 32 kDa, indicating successfulpurification of the protein. The β-lactamase activity of BlaC wasmeasured using nitrocefin (Calbiochem, MA, USA) as the substrate. Whennitrocefin is hydrolyzed by β-lactamase, a hydrolyzed product isproduced with a maximum absorption at 485 nm. Accordingly, the formationof the hydrolyzed product was monitored by absorbance at 486 nm (A₄₈₆)as an indicator of enzyme activity. Varying concentrations of thepurified protein (0.25 to 25 nM) were added to 1×MES buffer in a 96 wellplate, and the assay was initiated by the addition of 500 μM nitrocefin.The enzyme activity was monitored in EnVision® Multilabel reader (PerkinElmer, MA, USA) with intermittent shaking for a period of 30 minutes.The resulting absorbance of hydrolysis product resulting from thevarying concentrations of purified protein over time is illustrated inFIG. 1A. This demonstrates that enzyme activity is proportional to theconcentration of the concentration of the purified protein.

The observed rate of change in A₄₈₆ was converted to units of enzymeactivity (IM product formed per minute) by using the molar extinctioncoefficient of the hydrolyzed product (20,500 M⁻¹ cm⁻¹) and the pathlength of 0.29 cm. As illustrated in FIG. 1B, the enzyme activity wasfound to increase linearly as a function of enzyme concentration(R²=0.99), as expected. The purified BlaC was found to have an activityof 797±114 U/μM.

Generation of Anti-β-Lactamase (BlaC) Antibody

Antibody against purified protein was raised in rabbit (Bio SynthesisInc., TX, USA). Briefly the rabbits were immunized with purified proteinand a total of 100-150 ml of serum were collected with five boosts andfour bleeds. Eighth and 10th week bleeds were tested by ELISA and usedfor further experiments, as described below.

Western Blot Analysis

To determine whether the antibodies generated using the recombinant BlaCprotein could detect the protein in a biologically-relevant environment,a western blot analysis was conducted.

1) Sample Preparation

BlaC protein dilutions were made in phosphate buffered saline (PBS, pH7.4). 50 μL of the diluted protein was added to 450 μL of sputum sampleand mixed by pipetting. 500 μL of Transport Stabilization Solution(TSS), which primarily comprises MES buffer at pH 6.0, was added to thesample and further mixed to attain the best possible homogeneity. Toeach sample, 250 μL of Blue Sepharose™ 6 Fast Flow (Cibacron Blue)(washed and re-suspended in IX MES) (GE Health Care, Buckinghamshire,UK) was added and mixed again to obtain optimal albumin removal. VariousBlaC concentrations from 4 to 4000 ng were loaded onto the gels.

2) Detection

Ten μL of the BlaC samples (in sputum) were mixed with 10 μL of theloading dye, heated at 95° C. for 5 minutes and loaded onto 12% SDSpoly-acrylamide gels. 400 ng of BlaC (in PBS) was also loaded as apositive control. The gels were run in duplicates at a voltage of 150 Vuntil proper separation of the ladder was visually achieved. One of thetwo gels was coomassie stained to visually gauge protein levels andmigration. The other gel was transferred to a pre-treated PVDF membrane.Pre-treatment involved submersion in 100% methanol for 5 minutes oruntil the membrane was translucent, followed by equilibration in thetransfer buffer (3.03 g of Tris base, 14.4 g Glycine, 200 ml Methanol,and 800 ml ddH₂O) until the membrane no longer floated on the surface.The transferred membrane was immersed in blocking buffer (5 g non-fatmilk in 100 ml of PBS buffer) and blocked overnight at 4° C. on ashaking platform.

After overnight blocking, the blot was immersed in primary antibody(i.e., the polyclonal anti-BlaC antibody from rabbit) diluted 1:5000 inprimary antibody dilution buffer (i.e., wash buffer: 0.5 ml Tween 20 and1000 ml PBS buffer). The blot was incubated for 1 hour at 37° C. on ashaking platform. Incubation with primary antibody was followed bywashes with wash buffer (0.5 ml Tween 20 and 1000 ml PBS buffer) at roomtemperature for 10 minutes on a shaking platform. The washing step wasrepeated three times for a total of four washes. After the washes, theblot was immersed in secondary antibody (HRP-conjugated anti-rabbit IgG)diluted 1:10,000 in blocking buffer. The blot was incubated for 1 hourat 37° C. on a shaking platform. The blot was then washed again asdescribed above. The blot was processed with SperSignal® West PicoChemi-luminescent kit (Thermoscientific, IL, USA) according to themanufacturer's instructions and imaged after a 2 minute exposure.

Results: The western blot analysis demonstrated that BlaC concentrationsof 400 ng and above in sputum could be detected using the rabbitanti-BlaC antibodies (not shown). The concentration threshold fordetection could be attributed to the complexity of the sputum matrixobscuring detection of lower BlaC concentrations.

Enzyme-Linked Immunosorbent Assay (ELISA)

It is demonstrated above that western blot analysis using the generatedantibodies can detect BlaC protein in a biological sample (i.e.,sputum). To demonstrate the broader applicability of the anti-BlaCantibodies in other detection assays, an ELISA approach was explored.

One hundred μL of protein A/G (ProSpec, Ness-Ziona, Israel) (10 μg/ml incoating buffer: 1.59 g Na₂CO₃, 2.93 g NaHCO₃, 0.1 g Thimerosal, fill to1000 ml with ddH₂O) was dispensed in 96 well plates. The plates wereincubated at 4° C. overnight on a shaking platform. After the overnightincubation, the wells were washed with 200 μL of wash buffer (0.5% BSAand 0.05% Tween 20 in IX PBS) at room temperature on a shaking platformfor 5 minutes. The wash was repeated twice for a total of three washes.The wells were then blocked using the blocking buffer containing BSA (3%BSA in IX PBS) for 1 hour at 37° C. on a shaking platform.

BlaC samples were processed similar to the western samples with theexception that primary antibody (i.e., the rabbit polyclonal anti-BlaCantibody) was added at a concentration of 1:2500 into the sputum tofacilitate primary antibody binding with BlaC. The sample was incubatedfor 1 hour. 100 μL of sputum sample was dispensed into each well andincubated for 1 hour at 37° C. on a shaking platform. For bacterialsamples, BCG was incubated in 10 ml M-OADC-Tw at 37° C., 5% CO₂ until anOD₆₀₀ of 0.5 was achieved. Appropriate volume of this culture wascentrifuged and the pellet was re-suspended in 7H9 (BD Biosciences,California, USA). Bacterial suspension was further diluted in 7H9 mediaand instead of adding purified BlaC, bacterial dilutions were added tothe sputum and incubated for 4 hours. The samples were processed hereafter as in step 4.

After incubation with sputum samples, the wells were washed with washbuffer as described above and re-blocked with non-specific mouse IgGdiluted 1:5000 in blocking buffer to saturate the unbound protein A/G.The blocking was carried out for 1 hour at 37° C. on a shaking platform.After blocking, the wells were washed as described above. A 1:5000dilution of primary antibody (i.e., the rabbit polyclonal anti-BlaCantibody) was added to the wells in wash solution. The wells wereincubated for 1 hour at 37° C. on a shaking platform.

After incubation with primary antibody, the wells were washed as instep 1. 100 μL of secondary antibody (i.e., horseradish peroxidase(HRP)-conjugated goat anti-rabbit IgG) diluted in wash buffer wasdispensed in each well. The wells were incubated for 1 hour at 37° C. ona shaking platform. The wells were washed again as describe above and100 μL of 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)(ABTS) solution in citrate buffer was added to each well to serve as theHRP substrate. The plate was observed for development of color. Oncediscernible color was observed in the negative control (i.e., a wellwith no blocking buffer or secondary antibody) the reaction was stoppedusing 5% SDS. The formation of reduced ABTS as a measure of the amountHRP-labeled secondary antibody was read at 415 nm after approximatelyincubating for 5-10 minutes in EnVision® Multilabel reader (PerkinElmer, MA, USA).

Statistical analysis and data plotting was performed using Excel(Microsoft Corp., WA, USA). Standard deviations and p-values werecalculated.

Results: A standard plot of HRP activity was generated using BlaCdilutions in sputum. As illustrated in FIG. 2, the absorbance at 415 nm(i.e., indicating reduced ABTS) was found to linearly increase with theincrease in BlaC concentration in sputum (R²=0.87). The threshold fordetection of BlaC in sputum was calculated at 0.04 ng, beyond which thedetection was not linear and, hence, the absorption values were notincluded in the standard curve calculation.

Western Blot Analysis Using Bacteria

It is demonstrated above that standard western blot techniques usinganti-BlaC antibodies can detect the presence of BlaC in biologicalsamples (i.e., sputum). Accordingly, a preliminary assay was performedto ascertain whether whole bacteria are similarly detectable with thesame reagents and techniques.

A preliminary western blot was carried out with bacteria (i.e., M. bovis(BCG)) diluted in sputum using the general protocol described above.BlaC from bacterial dilutions could not be detected in the preliminarywestern blots, suggesting that the amount or specific configuration ofBlaC in live bacteria is not readily detectable by this basic method.The inability to detect BlaC from sputum sample by simple western blotanalysis could largely be due to the fact that BlaC is associated withthe bacterial cells, and a preliminary bacterial lysis might be requiredto obtain a more reliable estimate of BlaC presence. Prospectiveexperiments will examine lysed cells to better localize the proteinunder these conditions. The intensity and duration of lysis will bestandardized. Additionally the shortest incubation period of thebacteria in sputum resulting in reliable BlaC detection will beestablished. This refined assay will facilitate better understanding ofBlaC production in sputum and the utility of western blot analysis todetect tuberculosis in infected patients.

ELISA Analysis Using Bacteria

It is demonstrated above that ELISA techniques using anti-BlaCantibodies can detect the presence of BlaC in biological samples (i.e.,sputum). Accordingly, a preliminary ELISA was performed to ascertainwhether whole bacteria are similarly detectable with similar reagentsand techniques.

A series of dilutions of bacteria (i.e., M. bovis (BCG)) were generatedin sputum and assayed according to the general protocol described above.The absorbance values (i.e., indicating reduced ABTS and, thus, thepresence of HRP-labeled secondary antibody) for the various bacterialconcentrations sputum are illustrated in FIG. 3. A significant increasein signal was observed that correlated well with bacterial numbers. Thiscorrelation indicates the ability to detect whole bacteria in sputumusing this method. Ultimately, the present results demonstrate that theamount of BlaC was increasing with the increase in CFU, as expected.

However, it is noted that the levels of bacterial BlaC detected by ELISAwere low suggesting that the amount of BlaC produced less than theamount tested above to generate the standard curve. Thus, the presentresults were not extrapolated directly onto the current standard curve(as described above and illustrated in FIG. 2).

Surprisingly, the 10 CFU/well dilution of M. bovis (BCG) gave anunexpectedly high absorbance reading. This result is consistent with theinventor's observations that levels of BlaC activity are higher thanexpected when at low concentrations, such as 10 CFU of M. bovis (BCG) orMtb. These observations were measured using custom BlaC substrates.Overall, this is an approximately 100-fold increase in BlaC activityabove the expected level for this concentration, which is highlysignificant. Without being bound to any particular theory, theseobservations suggest that some aspect of the sputum environmentincreases the measurable BlaC activity, possibly as a result ofincreased expression, transport, activity or release of the protein. Allof these possibilities warrant further investigation to better controlfor expression and perhaps increase the sensitivity of BlaC diagnosticstrategies.

Conclusion

It is demonstrated that BlaC protein is detectible in sputum samplesusing antibodies generated against recombinant BlaC in rabbits. Theantibodies were demonstrated as useful diagnostic reagents when used inboth western blot analyses of sputum samples (incorporating theprotein), and ELISA-based analyses of sputum samples (incorporating theprotein or bacterial cultures).

II. The following describes an analysis of the β-lactamase DNA andprotein sequences and the design of a specific and sensitive detectionassay of tuberculosis-complex bacteria using β-lactamase nucleic acid asa biomarker.

Background

A key first step in the development of specific and sensitive detectiontechniques for any pathogen is identifying unique aspects (e.g.,sequences) that can reliably differentiate between the target pathogenand non-target organisms. Additionally, an ideal sequence target will bereliably expressed under a variety of conditions such as to provideample nucleic acid template for sensitive detection at all stages ofinfection.

As described herein, the blaC gene of tuberculosis-complex bacteria isdemonstrated to contain specific sequence domains that are not presentin any other bacterial species. These unique domains present theopportunity to generate very specific probes. Furthermore, there isextensive evidence that blaC is constitutively expressed intuberculosis-complex bacteria under nearly any condition at high levels,which makes it likely that detection reagents specific to the blaC RNAtranscript will be highly sensitive and applicable to nearly anycondition in vitro and during infections. Expanding upon published andavailable microarray and genomic data, analysis of the DNA and RNAsequences demonstrated that various sequences could be used forsensitive and specific reagents for tuberculosis-complex organisms. Forexample, TaqMan® and molecular beacon probes are appropriate approachesfor design of probes for this purpose. However, it will be understoodthat nearly any nucleotide recognition method could be used in a similarmanner using the regions identified herein.

Mycobacterium tuberculosis (Mtb) BlaC is a naturally occurring class-Aβ-lactamase (Flores, A. R., et. al., “Genetic analysis of theβ-lactamases of Mycobacterium tuberculosis and Mycobacterium smegmatisand susceptibility to β-lactam antibiotics,” Microbiology 151(2):521-532(2005), incorporated herein by reference in its entirety). BlaC ispresent in Mtb and all other tuberculosis-complex bacteria. The proteinconsists of 307 amino acids with a molecular weight of ˜32 kDa. BlaCconfers lactam antibiotic resistance to Mtb via hydrolysis of β-lactamantibiotics. As described below, use of the blaC RNA transcript isdemonstrated as useful a tool for Mtb diagnosis. The RNA transcript canbe amplified using reverse transcription, trapped, or directly amplifiedusing various RNA-RNA amplification systems, and the like. Moreover, thedescribed approaches could also be applied with very little modificationto detecting blaC in the Mtb chromosomal DNA. The primary modificationto the methodology for chromosomal detection is that DNA would beisolated and directly amplified or trapped for detection rather thanRNA. Moreover, the described techniques can be applied to other bacteriafrom the TB-complex.

Constitutive expression can help ensure that blaC-detection is alwayssensitive and quantifiable, especially in the conditions mimicking thehost environment prevalent during infection and latency in humans oranimals. Numerous microarray data accessed from available TB databases(NCBI-GEO, etc.) demonstrate that the blaC gene is expressedconstitutively when in intracellular environments, during growth in lab,and during infections, with no significant difference found betweensamples collected at 4 h and 24 h post-infection (Fontán, P., et al.,“Global transcriptional profile of Mycobacterium tuberculosis duringTHP-1 human macrophage infection,” Infection and Immunity 76(2):717-725(2008), incorporated herein by reference in its entirety), under aerobicconditions (Voskuil et al., “The response of mycobacterium tuberculosisto reactive oxygen and nitrogen species,” Front. Microbiol. 2:1-12(2011), incorporated herein by reference in its entirety), or during anoxidative stress response (Rodriguez, G. M., et al., “ideR, an essentialgene in Mycobacterium tuberculosis: role of IdeR in iron-dependent geneexpression, iron metabolism, and oxidative stress response,” Infectionand Immunity 70(7):3371-3381 (2002), incorporated herein by reference inits entirety). Although none of these previous studies specificallyexamined blaC according to the present evaluation of these data, theseobservations indicate that blaC transcription is constitutive and thegene is a valuable target for development of new methods for diagnosisof TB-complex bacteria. Furthermore, the blaC gene is constitutivelyexpressed 28 d post infection in experiments performed on BALB/c andSCID mice (Talaat, A. M., et al., “The Temporal expression profile ofMycobacterium tuberculosis infection in mice,” Proceedings of theNational Academy of Sciences of the United States of America101(13):4602-4607 (2004)) and at least 30 d post-inoculation in a Waynemodel depicting non-replicating persistence (Voskuil, M. I., et al.,“Mycobacterium tuberculosis gene expression during adaptation tostationary phase and low-oxygen dormancy,” Tuberculosis 84(3):218-227(2004), incorporated herein by reference in its entirety) making itconstitutively expressed under conditions commonly encountered by thesebacteria.

Accordingly, the amino acid sequences of the BlaC protein were comparedamong members of the TB-complex. The BlaC amino acid sequence from Mtbwas also compared against β-lactamases from other relevant pathogenicbacteria that are not part of the TB-complex. Similarly, the Mtb blaCgene sequence was compared with the β-lactamase gene sequences fromother relevant pathogenic bacteria that are not part of the TB-complex.Based on these comparisons, specific sequences that differentiated Mtband the TB-complex bacteria from other relevant pathogenic bacteria wereidentified and various detection reagents directed to the sequences weredesigned.

Demonstration of BlaC Specificity

The amino acid sequences of β-lactamases (BlaC) from bacteria belongingto the TB-complex, namely, M. tuberculosis, M. bovis, M. bovis (BCG), M.canettii and M. africanum, as well as β-lactamases (Bla) otherpathogenic bacteria were obtained from the NCBI database and alignedusing ClustalW2, a multiple sequence alignment tool from the EuropeanMolecular Biology Lab (EMBL). The β-lactamase (BlaC) protein showed 100%alignment identity within the TB-complex, indicating a high level ofconservation. FIG. 4 illustrates the relationship of β-lactamasenucleotide sequence between the species within the TB-complex and themost closely related Mycobacterium that is not within the TC-complex,i.e., Mycobacterium marinum. This comparison demonstrates that the M.marinum β-lactamase has a two node distance from the TB-complex,suggesting that even the mycobacterial most closely related to theTB-complex species has β-lactamase that is not closely related toTB-complex blaC sequence. Thus, strategies to differentiate Bla(β-lactamase from non-TB-complex species) from BlaC (β-lactamase fromTB-complex species) should be possible at the activity, protein andnucleotide level. FIG. 5 illustrates an alignment demonstrating thatBlaC amino acid sequence is highly conserved (with 100% identity) in allTB-complex bacteria (this consensus sequence is set forth in SEQ IDNO:2). Three motifs (I, II, and III) that are known to be important forenzymatic activity are indicated in the alignment.

In order to determine which sequences, if any, are unique to theTB-complex BlaC, the BlaC protein sequence from Mtb was compare to theBla protein sequences from various other bacteria using a multiplesequence alignment. This analysis, as illustrated in FIG. 6, revealedlow similarity at the protein level among the compared species. Even incomparison to other mycobacterial species, low sequence similarity wasobserved throughout the entire BlaC amino acid sequence. This suggeststhat specific detection of TB-complex by virtue of unique BlaC and/orb/ac sequence is possible, even in the presence of numerous naturalflora and other pathogens. The motifs that are conserved within theTB-complex bacteria and are necessary for the unique enzymatic activityare indicated in FIG. 6. The motif regions display the lowest amino acidsimilarity among the compared bacteria; there appears to be no sequencedBla that is closely related to that present in TB-complex. Inparticular, even the active site of BlaC contains several glycineresidues that are not present in any other bacterial species, indicatingthat even the active site sequences within the gene could be used ashighly specific probes for TB-complex bacteria in clinical orenvironmental samples. In particular, all three TB-complex BlaC motifs(I, II, and II) are part of the active site, as described in more detailin Wang, F. et al., “Crystal structure and activity studies of theMycobacterium tuberculosis β-lactamase reveal its critical role inresistance to β-lactam antibiotics,” Antimicrobial Agents andChemotherapy 50(8):2762-2771 (2006), incorporated herein by reference inits entirety. Briefly, the following discussion makes reference toresidues as numbered in the Wang reference with an indication of thecorresponding residue in SEQ ID NO:2, as disclosed herein. In all classA β-lactamases like BlaC, hydrolysis of the β-lactam substrates isachieved by a nucleophilic attack initiated by active-site serineresidue Ser70 (corresponding to the serine at position 84 of SEQ IDNO:2). It has been proposed that Glu166 (corresponding to the glutamicacid at position 182 of SEQ ID NO:2), a general base in the active site,is the serine70-activating residue. Glu166 (corresponding to theglutamic acid at position 182 of SEQ ID NO:2) has been established asbeing critical for the deacylation of the acyl-enzyme intermediate.Ser70 (corresponding to the serine at position 84 of SEQ ID NO:2) andLys73 (corresponding to the serine at position 87 of SEQ ID NO:2),located on α-helix H2 in the center of the active site are, also crucialand completely conserved in all class A β-lactamases. These residues aresurrounded by other important residues on β-strand B3 (Lys234, Thr235,and Thr237; corresponding to the lysine, threonine, and threonine atpositions 250, 251, and 253, respectively, of SEQ ID NO:2) and the loopregion between H5 and H6 (Ser130 and Gly132; corresponding to the serineand glycine at positions 142 and 144, respectively, of SEQ ID NO:2), aswell as the 2 loop (Glu166; corresponding to the glutamic acid atposition 182 of SEQ ID NO:2). These eight residues are all involved indirect hydrogen bonding interactions with β-lactam substrates. Two boundwater molecules, WAT36 and WAT65, are also highly conserved in thestructures of all class A β-lactamases determined to date. Use of activesite probes may have some advantage because divergence in this sequencewould potentially impact activity and would be selected againstevolutionarily, making the probe effective under most conditions.

Upon the discovery that there were numerous sequences within BlaC thatdiverge from the sequences of other Bla proteins, polynucleic acidprimers were considered for use in a detection system that wouldspecifically target TB-complex sequence. To this end, specific detailsregarding the nucleotide sequences of blaC gene were sought. As aninitial step in this process, an alignment was generated comparing thenucleotide sequences of the blaC gene from TB-complex bacteria with thesequences of the bla gene from a diverse set of organisms, includingother Mycobacteria sp. As illustrated in FIG. 7, similar results wereobtained with the nucleotide sequence as those using the proteinsequence. Specifically, while conserved regions were identified thatcould not be used for design of probes, several regions of blaC areunique to the TB-complex gene and are not present in any other bacterialspecies. These observations demonstrate that the design of probesspecific for TB-complex is possible, which probes can be used toevaluate the presence of these bacteria in nearly any clinical orenvironmental sample. The preferred parameters of probe design are thatthe probes are sensitive, specific and conserved throughout allTB-complex bacteria.

Design of blaC-Specific TaqMan® Probes

One embodiment of a PCR-based detection assay incorporates a TaqMan®probe, wherein the probe contains a fluorophore and a quencherconfigured such that when the probe is intact, no detectable signal isobserved. As is well-understood in the art, the probe is designed tohybridize to a portion of the target. The probe is cleaved when primersare extended from either side of the target region, thus releasing thefluorophore from proximity of the quencher and resulting in detectablesignal. Exemplary primer couples and probes were designed using EurofinsMWG Operon (AL, USA) software. Four primers and their respective probeswere selected based on nBLAST analyses, absence of secondary structure,melting temp (T_(m)) and length of the amplicon (Table 1). Primer probesets with a T_(m) difference of more than 10° C. were not considered.All probes, when subjected to BLAST, showed homology to the TB complexwith very low expected value (E-values), suggesting that the probabilityof their binding to any other bacterial DNA is highly unlikely.

TABLE 1Exemplary TaqMan primer and probe combinations designed for specificdetection of TB-complex blaC. The corresponding sequence identifiernumbers (i.e., SEQ ID NO:) are indicated in parentheses. Forward_primerReverse primer TaqMan ® probe 1 GACGAACGGGATACCACAACATCCAATCGGTGAGCAGTGC TTGCCGAGAACAAGCTGCTG (12) (21) CAACACCA (3) 2CATTCTGCTCCACGTTCAAG ATCGACCGAATGTCGTCAC TTTGTCCAGATGCGTGAGCG (13) (22)GGTTTTGGT (4) 3 CGAACGGGATACCACAACAC ATCCAATCGGTGAGCAGTGCTTGCCGAGAACAAGCTGCTG (14) (23) CAACACCA (5) 4 CATTCTGCTCCACGTTCAAGGTCGACCGAATGTCGTCACTG ATCAGTTGTCCAGATGCGT (15) (24) GAGCGGGTTTT (6)

Design of Molecular Beacon Probes

Another embodiment of a PCR-based detection assay incorporates amolecular beacon probe. As is well-understood in the art, the molecularbeacon probe contains a hairpin configuration with a loop comprising theprobe sequence that is complementary to the target nucleic acidsequence. The stem of the hairpin structure is formed by complementaryoligonucleotide sequences that are at the 5′ and 3′ end of the linearsequence. A fluorophore is covalently attached to the end of one of thestem oligonucleotides, whereas a quencher dye is covalently attached tothe end of the other stem oligonucleotide. When in the initial hairpinconfiguration, the probe does not emit a detectable signal. However,when annealing to the target sequence, the probe linearizes permitting asufficient distance to form between the fluorophore and quencher dyes,thus allowing a detectable signal. Such a probe can be incorporated intoPCR-based amplification assays. Exemplary molecular beacon probes weredesigned using BeaconDesigner™ 8.0 (Premier Biosoft, CA, USA). Becausethe use of beacon probes can also be incorporated into PCR-based assays,amplification probes were also designed in conjunction with specificbeacon probes (i.e., with forward and reverse primers that anneal 5′ and3′ to the annealing site of the beacon probe). The program facilitatesoptimal primer and beacon probe design with the annealing temperature ofthe beacon being at least 9° C. above the T_(m) of the primers. Fiveprimer and beacon probe sets were identified (Table 2). The beacons foreach set were subjected to nBLAST. The beacon showing the lowest E-valueis indicated (with *) in the table and will be the probe of choice forthis approach.

TABLE 2Exemplary molecular beacon primers and probes designed for specificdetection of TB-complex blaC, The corresponding sequence identifier numbers (i.e.,SEQ ID NO:) are indicated in parentheses. Forward primer Reverse primerProbe 1 CATCTGGACAA ATAGTGTATCG CGCGATCCCAGTGACGACATTCGGTGATCGCG (7)(16) (25) 2 TTCGCATTCTG ACTGGTGTAGG CGCGATCGCTCACGCATCTGGACAAGATCGCG (8)(17) (26) 3 TCGCATTCTG TGTCGTCACT CGCGATCCTCACGCATCTGGACAAACGATCGCG* (9)(18) (27) 4 TTCGCATTCTG ACTGGTGTAGGCGCGATCCAAAACCCGCTCACGCATGATCGCG (10) (19) (28) 5 ATACCACAACA GCCATCCAATCGCGATCGAACAAGCTGCTGCAACAGATCGCG (11) (20) (29)

Primers generated in this method, such as the disclosed primers, areextremely specific for TB-complex blaC nucleic acids, and are useful foreither RNA or DNA amplification and/or detection. In some detectionstrategies, RNA can be amplified without PCR. Moreover, both DNA andcDNA may be amplified randomly prior to PCR to increase sensitivity.Additional validation of these primer sets can involve examination oftheir conservation in all TB-complex bacteria and particularly thenumerous TB strains that have been sequenced to ensure that the finalprobe is specific, sensitive and highly conserved. With all three ofthese characteristics, a probe based on this strategy would beapplicable to all conditions. In contrast, without all three of thesecharacteristics, a probe based on this strategy would not be generallyapplicable to all conditions.

III. The following describes the successful use of nucleic acid primersand probes directed to the β-lactamase nucleic acid sequence tospecifically detect the presence of tuberculosis-complex bacteria.

Background

As described above in part II, β-lactamase (BlaC) can be a usefulbiomarker for tuberculosis-complex bacteria. As further described, insilico analysis of all available β-lactamase sequences demonstrated thatβ-lactamases are conserved in TB-complex bacteria, yet are unique ascompared to all other non-TB-complex sequences, including eukaryotes andprokaryotes. Accordingly, the present discussion validates the conceptthat TB-complex bacteria can be specifically detected by virtue of theunique nucleic acid sequence.

Method and Results

BeaconDesign 8.0 was used to design optimal primer and beacon sets withthe annealing temperature of the beacon being at least 9° C. above theT_(m) of the primers. Five primer and beacon sets were identified (seeTable 2, above). The beacon for each set was subjected to nBLAST, thatincludes all eukaryotes and prokaryotes currently sequenced for a totalof over 20 million sequences and >50 billion nucleotides as of Nov. 11,2013 (not shown). The beacon probe showing the lowest E-value andmaximum query coverage is SEQ ID NO:9 (Table 2, row 3), which was usedin conjunction with the primers with sequences set forth in SEQ IDNOS:18 and 27, as the probe of choice due to its lowest similarity tonon-mycobacterial sequences. Thus, probe 3 (set forth in SEQ ID NO:9)was the best of the five beacons designed using the BeaconDesignsoftware and was selected in conjunction with the corresponding primers(see Table 2, row 3) for synthesis, characterization, and validation indetection assays using chromosomal DNA from humans, Mtb and variousbacterial pathogens, including M. marinum, M. avium, Pseudomonas,Staphylococcus, and others. The selected probe and primer set wereordered from Integrated DNA technologies and used for the real-timeexperiments.

Mycobacterial DNA was extracted from BCG cultured in 100 ml M-OADC-Twmedia at 37° C. until an OD₆₀₀ of 0.8 was achieved. The cells werepelleted out by centrifugation at 3500 rpm for 15 min and re-suspendedin minimal amount of saline solution (0.9%). Cell lysis was achieved byaddition of 500 μL of acid washed beads in 750 μL of TE buffer per 250μL of re-suspended pellet, followed by vortexing. Lysed cells werecentrifuged at 10,000 rpm for 10 min and the supernatant was collected.RNAseA was added to the supernatant to a final concentration of 0.1mg/mL followed by incubation at 37° C. for 1 hr in order to eliminateRNA contamination from the supernatant. Equal volumes ofphenol-chloroform-isoamyl alcohol was added to the supernatant andincubated for four minutes with gentle rocking followed bycentrifugation at 10,000 rpm for 10 min. The supernatant was collectedand DNA was precipitated by adding 3 M NaOAc in the ratio of 1:10 andtwo volumes of cold ethanol. The precipitated DNA was then pelleted outby high speed centrifugation (13,000 rpm) followed by 70% ethanolwashes. The DNA pellet was dissolved in TE and stored at 4° C. forfurther use. DNA concentration and quality of the extracted DNA wasdetermined.

The concentration of extracted Mycobacterial DNA was estimated to be 455ng/μl. 1:10 dilution of this sample was loaded onto an agarose gel tovisually establish DNA quality (not shown).

Molecular Beacon real-time PCR was carried out using the selected set ofprimers and probe (see Table 2, row 3). The PCR reaction contained 10 to100 ng of BCG DNA, 7 mmol/L final concentration of MgCl₂, 200 μmol/Lconcentration of dNTP, 2.5 U of taq DNA polymerase, 0.2 μmol/L finalconcentrations of the primer pair and 0.2 μmol/L final concentration ofthe beacon. The PCR was performed on the Applied Biosystem, step onereal-time PCR system, using the following cycling conditions: 95° C. for3 min, followed by 50 cycles of 94° C. for 20 s, 42° C. for 20 s, and72° C. for 20 s. Each sample was loaded as duplicates for technicalcontrols.

Real-time amplification using the beacon was carried out and the resultsare summarized in Table 3. A minimum of 10 ng of DNA per well wasdetected, which was the genomic equivalent of 2×10⁶ Mycobacterium cells.Representative amplification plot for 10 ng and 50 ng DNA per well havebeen shown in FIG. 8.

TABLE 3 Cycle number at which detection was possible for variousconcentrations of DNA DNA concentration (ng) Cycle number^(a) 10 35 ±0.04 50 37 ± 0.3  100 ND^(b) ^(a)Number of cycles at which thefluorescence generated by the cleavage of the beacon hairpin due toamplification by the primer set was detectable above the background.^(b)Not detected

Additionally, RNA was extracted and cDNA was synthesized from variousbacterial samples including MRSA, E. coli, Salmonella, Mycobacteriumsmegmatis, Pseudomonas PAO1 and Bacillus. Briefly, the bacterial cellswere lysed using TRIzol reagent (Invitrogen). RNA samples were treatedwith RNase-free DNase I (Promega), followed by purification using theRNeasy Mini Kit (Qiagen). The concentration of RNA was estimated using aNanoDrop ND-1000 spectrophotometer (version 3.1.0; Thermo FisherScientific). Reverse-transcription reactions on total RNA were performedusing the First Strand cDNA Synthesis Kit (Invitrogen) with randomdecamers.

Real-time PCR was carried out using the synthesized cDNA and thedesigned Beacon. No signal was observed with cDNA corresponding to thevarious bacteria (not shown), suggesting that the designed probes asexpected were specific for the mycobacterial complex.

To further improve the threshold for detection (e.g., to facilitatereliable and specific detection of lower amounts of TB-complex bacteriain a sample), bacterial genomic DNA yields can be enhanced throughachieving enhanced bacterial cell lysis. Furthermore, an improveddetection threshold can be achieved through enhanced DNA isolation andpurification techniques. This is a useful improvement and considerationthe observation that DNA concentrations higher than 50 ng did not giveprovide signal (not shown). This result implied that there was aconsiderable amount of impurity in the extracted DNA sample and athigher concentrations these impurities inhibited the real-time PCRassay.

Conclusion

It is demonstrated that nucleic acid probes and primers designed tohybridize to unique sequences of consensus blac from TB-complex bacteriafacilitates the PCR-based specific detection of TB-complex bacteria anddoes not cross-react with other, non-TB-complex bacteria.

IV. The following describes the generation and purification of goatpolyclonal antibodies that bind to BlaC protein. The polyclonalantibodies were analyzed for utility as either a capture or detectionreagent in a lateral flow detection platform.

Antibody Production

Polyclonal Goat anti-BlaC (PAC 8577) was custom made by PacificImmunology. Briefly, pre-immune serum was first collected and then 200μg of BlaC antigen and 200 μg of Complete Freund's Adjuvant wereinjected into a goat host. Five subsequent boosts of 100 μg of antigenand 100 μg of Incomplete Freund's Adjuvant took place over the next sixmonths. About two months from the initial immunization, the firstproduction bleed commenced, collecting approximately 300 ml of serum. AnELISA was performed on the first bleed with a titer of ˜1:500,000. Thesubsequent production bleeds occurred about every two weeks thereafter.The second production bleed produced a serum volume of 329 ml. The thirdproduction bleed produced a serum volume of 250 ml. The serum was splitinto two batches and affinity purified separately to ensure isolation ofall the specific antibody from the serum. Approximately 16.1 mg ofaffinity purified antibody were obtained and the ELISA results showed atiter of ˜1:500,000. The fourth production bleed produced a serum volumeof volume of 290 ml. The fifth production bleed (performed after afour-week interval) produced a serum volume of 310 ml. This bleed wasalso affinity purified, following the same procedure described above.Approximately 15.4 mg of purified antibody was obtained and the ELISAresults showed a titer of ˜1:500,000. The sixth production bleedproduced a serum volume of 292 ml. This bleed was also affinitypurified, using BlaC immobilized on an affinity support column.Approximately 14.4 mg of purified antibody was obtained and the ELISAresults showed a titer of ˜1:500,000.

Affinity purification referred to above was generally conducted bychemically immobilizing BlaC antigens on an affinity support column.When the goat serum was passed over the column, goat anti-BlaCantibodies bound to the BlaC antigens. The support was washed withadditional buffer to remove non-bound components. Finally, elutionbuffer was added to remove goat anti-BlaC from the immobilized BlaC onthe support, resulting in the releasing of goat anti-BlaC antibodies inits purified form from the original serum.

ELISA titer results represent a relative measurement of the quantity ofpeptide-specific antibody present in serum samples. Titer was determinedby diluting the serum samples until the antibody level gave values onthe plate reader approaching background levels. Titer of 1:500,000 meansthis is the dilution that gives a reading of approximately 0.1 O.D.above background.

Lateral Flow Assay

A preliminary BlaC Lateral Flow Assay was performed using the polyclonalGoat anti-BlaC antibody 0.7 mg/ml (PAC 8577) as both capture antibody onthe test line and label antibody in the gold conjugate. The test linewas striped at 0.5 mg/ml on nitrocellulose membranes. The Goat anti-BlaCantibody was conjugated to 40 nm colloidal gold particles at 6 μg per 1ml of gold. Buffers and blocking agents were optimized for the detectionof BlaC spiked in a buffer solution. At this scale, the limit ofdetection was 10 ng/ml of BlaC at 15 min. The preliminary lateral flowassay demonstrates excellent sensitivity, establishing that that thegoat polyclonal antibody reagent can be useful in capture and detectionof BlaC. Ideally, the polyclonal reagent is paired with anotherpolyclonal antibody or specific monoclonal anti-BlaC antibody to provideexcellent specificity and sensitivity in an immunoassay.

V. The following is an overview describing the successful production andanalysis of monoclonal antibodies from rabbit (“RabMAb®”) thatspecifically bind to the active BlaC enzyme of TB-complex bacteria.

Overview

Active and inactive forms of BlaC protein antigens were recombinantlyproduced as described above and contained His-tags to facilitateisolation/purification. The active form of BlaC is also referred toherein as KES-1A antigen, whereas the inactive antigen, referred toherein as KES-1B, is a BlaC preparation with a high proportion of enzymedemonstrating diminished enzymatic activity). KES-1B and an irrelevantHis-tagged protein, referred to herein as KES-1C antigen, were used forcounterscreening.

Two, 3-month old New Zealand White rabbits were subject to apre-immunization bleed (5 mL) one day prior to immunization. The rabbitswere immunized using a customized protocol of four subcutaneousinjections and two production bleeds per rabbit. The first immunizationincluded the KES-1A antigen aliquot (0.4 mg) combined with CompleteFreund's Adjuvant (CFA). The subsequent injections included 0.2 mgKES-1A with incomplete Freund's Adjuvant (IFA) at 3, 5, and 7 weeksafter the initial injection. The production bleeds were performed justbefore the last injection and at two months after the initial injection.

Serum obtained from the production bleeds were screened by ELISA toestablish titers against the KES-1A (active BlaC), KES-1B (inactiveBlaC), and KES-1C (irrelevant protein) antigens using Abeam standardprotocols. The immunization produced good antibody titers (not shown)for the immunogen was 1:64,000. The cross-screening demonstrated verylittle cross-reactivity for the KES-1C (His-tagged) antigen, indicatingminimal reactivity with the His-tag, and an overall lower titer againstthe “inactive” BlaC (his-tagged).

The rabbit with higher anti-BlaC titer was selected for splenectomy andmonoclonal development. The rabbit was subject to a final immunogenboost and the spleen was harvested and splenocytes were isolated usingstandard protocols four days thereafter. Two hundred million lymphocytecells were fused with 100 million fusion partner cells on two separatedays and plated on twenty 96-well plates on each day. The plates werekept in tissue culture incubators under standard conditions. Cell growthwas examined 2-3 weeks after fusion and fusion efficiency (calculated asthe total number of wells containing hybridoma cell colonies divided bythe total number of wells examined) was analyzed. A minimum of twoplates were examined per fusion.

After fusion, all multiclone supernatants were screened by standardELISA for reactivity with the BlaC protein (KES-1A) antigen. For eachELISA screen, antigens were coated at 50 ng per well, and the originalrabbit bleed at 1:10,000 dilution was used as a positive control. Atotal of 29 ELISA positive multiclones were identified and then expandedto 24-well plates. A confirming screen was subsequently performed forall positive multiclones using the BlaC protein antigen (KES-1A), theinactive BlaC (KES-B), and the irrelevant his-tagged protein antigen(KES-1C), as described above. Fifteen multiclones were determined toshow reactivity with the BlaC protein (KES-1A) antigen, and thereforewere confirmed positive.

After the multiclone supernatant evaluation, three clones (i.e., 20, 22and 27) were selected for subcloning. Subcloning was performed bylimited cell dilution method, where cells were seeded into 96-wellplates at a concentration of 1 cell/well, with a total of three 96-wellplates per multiclone. All subclones were screened again by ELISAagainst the BlaC protein antigen. For each multiclone, 12 positivesubclones were selected and expanded into 24-well plates. A subcloneconfirming ELISA screen was performed using the BlaC protein (KES-1A)antigen, the inactive BlaC (KES-1B) antigen, and the irrelevanthis-tagged protein (KES-1C) antigen on the 12 positive subclonesupernatants per multiclone (36 subclones total). The IgG concentrationof the 36 subclone supernatants was measured by ELISA as well. All 36subclone supernatants showed positive reactivity towards the BlaCprotein antigen.

A competition ELISA was performed to better simulate antibody bindingunder similar conditions as during the lateral flow assay. For thecompetition ELISA, the plate was coated with the BlaC protein (KES-1A)antigen, and was run using a standard ELISA protocol except the subclonesupernatants were incubated with either PBS, or the antigen for 1 hourin solution before being added to the antigen-coated plate. This is tobetter simulate how the antibody would bind the antigen in solution in alateral flow assay. Subclones from parental clones 27 and 22 showeddecreased binding to the plate when incubated with the BlaC protein(KES-1A) antigen in solution first, which indicates that these clonesbind strongly to the antigen in solution. Subclones (e.g., 20-8, 22-12,and 27-11) were selected to be expanded and frozen according to theprotocols described below.

The hybridoma complete growth medium included RPMI 1640 medium with 0.05mM 2-mercaptoethanol, Abeam Media Supplement A (catalog numberab138912), containing antibiotic/antimycotic/Gentamicin), and 10% fetalbovine serum.

Hybrodima propagation was performed by centrifugation with subsequentresuspension in hybridoma complete growth medium. New cultures wereestablished at 0.1 million cells/ml and maintained between 0.1-1 millioncells/ml. Medium was changed every 3 to 6 days. Subclones were screenedand determined to be negative for Mycoplasma.

Hybridoma cells were frozen in 90% FBS and 10% DMSO.

Monoclonal antibody production is performed by expanding the selectedsubclones and harvesting supernatants, as is commonly performed in theart. Purified monoclonal antibodies can be tested for IgG concentration,specific activity by ELISA, and purity by SDS-PAGE.

Conclusion

RabMAbs® were successfully generated against the BlaC protein. Furtherefforts include production of purified antibodies and screening forspecific detection of BlaC protein from TB-complex bacteria as comparedto the BlaC protein from non-TB-complex bacteria, the characterizationof the specific epitope conferring specific recognition, and theimplementation of the monoclonal antibodies in detection assay formats.

VI. The following is an overview describing the successful productionand analysis of monoclonal antibodies from mouse that specifically bindto the active BlaC enzyme of TB-complex bacteria.

Monoclonal antibodies were produced in mice that specifically bind toBlaC as compared to an irrelevant protein (TEM1). The antibodies weregenerated and were screened by ELISA according to the same generalstrategies described above in Part V for Three clones (i.e., 31A, G1 andH1) produced ample amounts of antibody that exhibited nocross-reactivity with the irrelevant TEM1 antigen, even with TEM1 atdouble concentration (1.0 μg/ml) when compare to BlaC 0.5 μg/ml antigen.

Accordingly, Mouse monoclonal antibodies were successfully produced thatspecifically bind to BlaC. Further efforts include production ofpurified antibodies and screening for specific detection of BlaC proteinfrom TB-complex bacteria as compared to the BlaC protein fromnon-TB-complex bacteria, the characterization of the specific epitopeconferring specific recognition, and the implementation of themonoclonal antibodies in detection assay formats.

VII. The following describes the successful detection of BlaC present insputum using a lateral flow assay format incorporating therabbit-derived monoclonal antibodies described above.

Methods

The lateral flow assay components were prepared and assembled accordingto the following procedures.

Nitrocellulose Membrane Preparation: Test and control lines were sprayedon Sartorius CN 95; 30 mm with 1.0 mg/ml antibody (Mouse anti-BlaC H-1,RabMab 20-8, RabMab 22-12 or RabMab 27-11) or 0.5 mg/ml Goat anti-BlaCas the test line and 0.5 mg/mL of Goat anti-Mouse, Goat anti-Rabbit,Donkey anti-Goat or the combination of three as the control line.Striping Buffer is IX PBS pH 7.4; 0.2% Sucrose. The test line andcontrol line were sprayed 7 mm apart using the Biodot sprayer. The testline was 11 mm from the bottom of the membrane. Membranes were stripedat a rate of 1.0 μl/cm. The membranes were dried at 37° C. for 1.0 hourand stored in a desiccated foil pouch. Striped membranes were keptdesiccated overnight before blocking.

Antibody Gold Conjugation Protocol: Using Slide A-Lyzer 10000 MWCORabMab 20-8 and Mouse anti-BlaC H-1 were dialyzed in 10 mM PotassiumPhosphate pH 7.4 overnight. After dialyzing, the final concentration ofRabMab 20-8 was 1.0 mg/ml, and Mouse anti-BlaC H-1 was 0.715 mg/ml.Amicon Ultra-0.5 Centrifugal Filter devices were used to concentrate anddialyze Goat anti-BlaC, RabMab 22-12 and RabMab 27-11. The finalconcentration of Goat anti-BlaC was 13.68 mg/ml; RabMab 22-12 had theconcentration of 4.48 mg/ml, and RabMab 27-11 had 3.84 mg/ml.

Colloidal Gold Solution, at room temperature, was adjusted to desirablepH for each antibody (pH 7.6 for RabMab 22-12, 8.0 for Goat anti-BlaC,8.4 for RabMab 20-8, 8.6 for RabMab 27-11 and Mouse anti-BlaC) withfresh made 0.1M K₂CO₃. Then, the dialyzed antibodies were added tocolloidal gold solution with vortexing. The solution was incubated for30 minutes on a rotator at room temperature. The conjugate was blockedwith 10 μl (for every 1 ml of OD 2 colloidal gold) of Conjugate BlockingBuffer (25 mM Borate Buffer; 6% BSA; 0.2% Bioterge; 0.3% Sucrose) on arotator at room temperature for 10 minutes. The gold conjugate wascentrifuged at 12000 RPM, 4′C for 20 minutes and the supernatantdiscarded. The conjugate pellet was re-suspended with 0.2 ml (for every1 ml of OD 2 colloidal gold) Conjugate Re-suspension Buffer (25 mMBorate Buffer; 1.2% BSA; 0.04% Bioterge; 0.06% Sucrose). ConjugateBlocking Buffer and Conjugate Re-suspension Buffer with pH 7.8 wereadded to gold conjugate solution pH 7.6 and 8.0: those with pH 8.6 wereadded to gold solution pH 8.6. OD of gold conjugate was checked using aspectrophotometer and adjusted to 10-12. The gold conjugate was storedat 4′C until use.

Membrane blocking: Striped membrane CN 95 was placed into Lateral FlowBlocking solution (25 mM KP04; 0.2% Casein; 0.5% Boric Acid; 0.02%Sucrose; 0.1% Surfactant 10-G; 0.5% PVA) with the orientation of thetest line at the bottom of the nitrocellulose and the control line onthe top of the nitrocellulose. The blocking solution was allowed to wickup to the top of the membrane. The membrane was removed from theblocking solution and placed in a finger rack to dry at 37° C. for 1hour. Blocked membranes were placed in a desiccated plastic bag andstore in a dry room.

Glass fiber blocking: 300 mm Millipore G041 glass fibers were saturatedwith LF blocking buffer using a P-1000 pipette. After 15 minutes, thefibers were transferred to a paper towel. After one minute, the fiberswere place on the finger rack to dry at 37° C. for an hour. Blockedglass fibers were put in a plastic bag with desiccators and store in adry room.

Gold Conjugate Pad Preparation (for dried conjugate pad testing method):The OD (10 to 12) gold conjugate was prepared by adding 10% Sucrose and5% Trehalose to the conjugate. The gold conjugate was dispensed bypipetting at the rate of 1 μL/mm on 4 mm assembled test strips. The teststrips were dried at 37° C. for 1 hour, packed in a desiccated foilpouch, and stored in a dry room.

Test Strip Lamination: Striped nitrocellulose membrane was laminatedonto vinyl backing card. The wick pad was placed on the top portion ofbacking overlapping the membrane by 2 mm. The 10 mm conjugate pad wasoverlapped onto the membrane by 2 mm. The sample pad was placed on topof the conjugate pad with a 15 mm overlap from the bottom of backingcard.

Test Strip Cutting: Assembled cards were cut into 4 mm strips usingBiodot CM4000 cutter.

The BlaC lateral flow assay was tested according to the followingprocedures.

General assay testing: 70 μL of running buffer (negative control) ordiluted sample in running buffer (positive control) was pipetted intosample pad. The running buffer contained 1×PBS: 0.05% BSA; 0.1% TritonX-100; 0.2% Tween-20. The test line intensity after the 15th minute wasobserved and evaluated.

Wet testing method: 3 μL of gold conjugate and 30 μL of sample or buffer(negative control) was pipetted into a well of a micro plate and mixed.Test strips that did not have conjugate and sample pads were allowed tosit in the well for 15 minutes. The test line intensity was observed andevaluated.

Dried down conjugate method: Pipetted 4 μL of gold conjugate ontoconjugate pad of each strip. Dried the strips at 37° C. for 1 hour.

Antibody pairing: Each antibody was striped on CN95 membrane and goldconjugation was performed. Each antibody was experimented (paired)against itself and other antibodies using the generated membranes andgold conjugates.

Results

To determine optimal pH for the new anti-BlaC RabMAb® antibodies, thegold conjugation protocol was performed using a pH range of colloidalgold from 7.2 to 9.0, with 0.2 of increment. For each pH gold conjugatesolution, the concentration of antibodies was 6 μg/ml. 5.0 μL of eachfinal gold conjugate solution was spotted on G041 glass fiber and driedat 37° C. for 30 minutes.

Generally, the optimal pH of a gold conjugate would be the lowest pHthat, after drying, exhibits the best cherry red color on the glassfiber. This indicates there is little to no aggregation of thegold-antibody conjugate. The optimal pH of the various antibodies wereobserved to be pH 8.4 (RabMab 20-8), pH 7.6 (RabMab 22-12), and pH 8.6(RabMab 27-11).

Antibody pairs were screened among Goat anti-BlaC, Mouse anti-BlaC H1,RabMab 20-8, RabMab 22-12, RabMab 27-11, to determine which pair(s)yield(s) useful signal intensities. All pair combinations wereperformed, such that each pair member served as a detection antibody(i.e., gold-conjugated) and a capture antibody (striped on a “test line”(TL). The gold conjugation protocol described above was performed toprepare each antibody. The capture antibodies were striped on CN95membrane. For control, 0.5 mg/ml Donkey anti-Goat, Goat anti-Rabbit,Goat anti-Mouse, or combinations of these control capture antibodieswere striped on a control line (CL). Membranes were blocked and BlaC wasapplied in the wet testing method (described above).

Fifteen pairs of antibodies yield observed signals on test lines (TL).Criteria of determining good pair(s) of antibodies included: negativecontrol stays negative (no TL observed); 50 ng/ml and 3 ng/ml BlaC havestrong signal intensities. A pair of Goat anti-BlaC as a capture andRabMab 22-12 as a conjugate meets the criteria. See FIG. 9. Asillustrated in FIG. 9, the negative control remained negative (leftstrip), the signal intensity of 50 ng/ml BlaC (middle strip, lower band)is 7 and that of 3 ng/ml BlaC is shadow (right strip, lower band).Another pair of antibodies matching the criteria is RabMab 20-8 as acapture and RabMab 27-11 as a conjugate. See FIG. 10, which alsoillustrates no lines for the negative control, the 50 ng/ml BlaC is 7 ofthe signal intensity (middle strip, lower band), and 3 ng/ml BlaC showsshadows of that signal (right strip, lower band).

General observations include that the strips had pinkish backgroundswith some exhibiting aggregation at the bottom. This was attributed tothe wet method not containing 5% Trehalose and 10% Sucrose to stabilizeand flow smoothly on test strips.

For negative control, weak test line signals (the intensity from +/−toVF) were observed on some strips. Furthermore, test strips withoutconjugate and sample pads do not eliminate non-specific binding verywell; as a result, some feint signals were observed.

Suboptimal coloring was also observed in some cases where bold red linesat the control line (CL) position were not observed because antibodiesin the gold-conjugate solutions were not compatible or did not bind wellto antibodies at test line (TL) position. For example, where the RabMab22-12 antibody served as the gold conjugate and was paired with Goatanti-Rabbit antibody was striped on the on control line (CL), thepositive control signal was weak. Although these two antibodies arecompatible binding partners, they did not bind well together as would beexpected. In other combinations, the gold-conjugated antibody (e.g.,Goat anti-BlaC) was paired with an incompatible antibody on the controlline (CL), such as Goat anti-Rabbit (instead of the control Donkeyanti-Goat). Because these were not compatible antibodies, a CL signalwas not a bold red line. However, in cases using Mouse anti-BlaC H-1,and the three RabMab antibodies, optimal control coloring was observedbecause the control lines consisted of both Goat anti-Mouse and Goatanti-Rabbit antibodies.

Cross reactivity to other β-lactamase (Bla) proteins was evaluated forRabMab 22-12 and RabMab 27-11 (serving as the gold-conjugated detectionantibody). RabMab 22-12 was paired with goat anti-BlaC as the captureantibody and RabMab 27-11 was paired with RabMab 20-8 as the captureantibody and the test line (TL). Gold conjugation, membrane striping,blocking, and dried down testing were performed as described above usingBlaC and five difference β-lactamase (Bla) proteins (at 1 μg/ml and 500ng/ml).

Only BlaC-positive control samples yielded positive results at test line(TL) positions, while the other β-lactamase samples did not producepositive signals (not shown). Therefore, no cross activity occurredbetween the two antibody pairs (Goat anti-BlaC/RabMab 22-12 and RabMab20-8/RabMab 27-11) and four different i-lactamases (AG Scientific LN:1163, AG Scientific LN: 2467, Novus Biological and OXA-48).

It is noted that the gold conjugate solutions for dried down conjugatemethod consisted of 5% Trehalose and 10% Sucrose. As a result, theconjugates stabilized and flowed smoothly on test strips. Thus, allstrips had clear backgrounds, and did not exhibit any precipitatedparticles during test running. In addition, the control lines (CL) oftest strips for pairing of Goat anti-BlaC (capture) and RabMab 22-12(conjugated detection) antibodies were bold red lines. However, thecontrol lines from the pairing of RabMab 20-8 (capture) and RabMab 27-11(conjugated detection) antibodies were fade red lines because RabMab27-11 did not bind well with Goat anti-Rabbit antibody used at thecontrol line (CL). A weak control line notwithstanding, the red controlline indicates the strip works properly.

The negative control samples remained negative because the test stripswere assembled with conjugate and sample pads such that non-specificbinding was not observed.

Signal intensity was assessed for chosen antibody pairs with a series ofBlaC concentrations. Gold conjugation, striping blocking, and dried downtesting method protocols were generally followed as described above forincreasing concentrations of BlaC.

The pairing of RabMab 20-8 (capture) and RabMab 27-11 (conjugateddetection) antibodies detected the presence of BlaC at the concentrationof 9 ng/ml (not shown). The pairing of Goat anti-BlaC (capture) andRabMab 22-12 (conjugated detection) antibodies detected the presence ofBlaC at the concentration of 3 ng/ml.

It is noted that the gold conjugate solutions for dried down conjugatemethod consisted of 5% Trehalose and 10% Sucrose. As a result, theconjugates stabilized and flowed smoothly on test strips. Thus, allstrips had clear backgrounds, and did not exhibit any precipitatedparticles during test running. In addition, the control lines of teststrips from the pairing of Goat anti-BlaC (capture) and RabMab 22-12were bold red lines. However, the control lines from the pairing ofRabMab 20-8 (capture) and RabMab 27-11 (conjugated detection) were fadedred lines because RabMab 27-11 did not bind well with Goat anti-Rabbitantibody, which was striped at the control line (CL). A weak controlline notwithstanding, the red control line indicates the strip worksproperly.

Negative control samples remained negative because test strips wereassembled with conjugate and sample pads, such that non-specific bindingwas not observed.

Discussion

Regarding antibody pair screening, the two best antibody pairs from atotal 25 pairs were Goat anti-BlaC as a capture reagent paired withRabMab 22-12 as a conjugated detection reagent, and RabMab 20-8 as acapture reagent paired with RabMab 27-11 as a conjugated detectionreagent. RabMab 20-8, RabMab 22-12, RabMab 27-11 and Mouse anti-BlaC H-1did not pair to themselves. RabMab 22-12 did not pair well with RabMab27-11, RabMab 22-12 and RabMab 27-11 (as capture antibodies) did notpair well with Mouse anti-BlaC H-1 and RabMab 20-8 (as conjugateddetection antibodies), respectively.

Regarding cross reactivity, Goat anti-BlaC/RabMab 22-12 and RabMab20-8/RabMab 27-11 pair did not exhibit any cross activities withβ-lactamases from other, non-TB complex sources (AG Scientific LN: 1163,AG Scientific LN: 2467, Novus Biological and OXA-48).

Regarding signal intensity, the pair of RabMab 20-8 and RabMab 22-12detects the presence of BlaC at the concentration of 9 ng/ml. The pairof Goat anti-BlaC and RabMab 27-11 detects the presence of BlaC at theconcentration of 3 ng/ml. The test strip format demonstrated thecapacity for optimization to produce clear assay background andminimized (or no) precipitated detection particles.

Accordingly, these results demonstrated the successful development oflateral flow assays incorporating pairs of anti-BlaC antibodies thatdemonstrate accurate and sensitive detection of BlaC markers in arelevant biological sample.

VIII. The following describes an additional embodiment of a lateral flowassay that was successful in detecting BlaC in sputum.

Methods

Assay strips were assembled. As a preliminary step, detection reagentwas generated by generating antibody-gold conjugates, as describedabove, using 6 μg/mL with the RabMab 27-11 clone at pH 8.6. Theconjugate sample was adjusted to an OD of 10 and 10% sucrose and 5%trehalose were added. The gold conjugate was striped onto G041 10 mmglass fiber conjugate pad (Millipore) and blocked with LF blockingbuffer) at 12 μL/cm. The pad was dried at 37° C. for 1 hour and keptdesiccated overnight before use. Membrane striping was performed on CN95nitrocellulose membrane using Goat anti-Rabbit (Lampire Biological Labs)and Donkey anti-Goat (Lampire Biological Labs; LN: 09C21103) antibodiesin a final concentration of 1 mg/mL. Test line was striped withGoat-anti BlaC at a concentration of 1 mg/mL (the antibody wasconcentrated from 0.7 mg/mL to 13.5 mg/mL before striping). The membranewas dried at 37° C. for 1 hour and kept desiccated overnight before use.The CN95 membrane, wick pad (EMI 30250), and conjugate pad (G041), wereassembled onto a backing card (G&L; PN: GL-57065; LN: 1100603/1030805)and cut into 4 mm wide strips. Such assemblies (assay strips) could beused immediately or stored in desiccated environment for long term.

For sample preparation, 50 mL of digestion buffer (0.2 M citrate, 50 mMTCEP, 0.3% fish gelatin, 0.1% Proclin, pH 6.0) was prepared.Specifically, 2.941 g of citric acid and 0.716 g were added to about 40mL of DI water and stirred until completely dissolved. 1.5 mL of 10%fish gelatin and 0.515 mL of Proclin 950 were added to the solution. ThepH was titrated up to 6.0 with 10 N NaOH and QS to 50 mL with DI waterand with final pH confirmed at RT.

3 mL of digestion buffer was added to 3 g sputum (1:1 ratio). The samplewas vortexed and allowed to digest for 30 minutes. The sample wasfiltered through a 5 um filter (Whatman 5 um GD/X Filter, Cat #6870-2550(25 mm diameter)) and filtrate was collected.

BlaC was spiked in sputum sample at final concentrations of 0.5 ng/mL, 1ng/mL, 2 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL, 200ng/mL, 500 ng/mL, and 1000 ng/mL. The remainder of the unspiked sputumfiltrate was filtered through a 0.2 um filter (Whatman 0.2 um GD/XFilter, Cat #6870-1302 (13 mm diameter)). The filtrate was collected andspiked with BlaC at the concentrations indicated above for thesingle-pass filtrate. 100 uL of samples were pipetted onto each well onthe 96-microplate plate. Individual strips were dipped into the samplewells and stopped running after 15 minutes by removing wick pad,conjugate pad, and sample pad from strips. The strips were inserted intoan Axxin reader (Axxin, Fairfield, Australia) for quantitation.

The CL and TL intensities from the Axxin quantitation for the 5 μmsputum filtrate spiked with varying concentrations of BlaC are providedin Table 23. The TL readings are illustrated graphically in FIG. 37. TheCL and TL intensities from the Axxin quantitation for the 0.2 μm sputumfiltrate spiked with varying concentrations of BlaC are provided inTable 24. The TL readings are illustrated graphically in FIG. 38.

Results

The intensities of control line (CL) and test line (TL) signals asdetermined by the Axxin reader are set forth in Tables 4 and 5 for thesputum samples filtered with 5 μm (first pass) and 5 μm followed by 0.2μm (second pass) membranes, respectively. The TL intensities obtainedfrom each filtrate are also graphically illustrated in FIGS. 11A and11B.

TABLE 4 5 μm sputum filtrate (first pass) readings spiked with varyingconcentrations of BlaC Concentration CL Intensity TL Intensity Negative12890 472 0.5 ng/mL  12905 428  1 ng/mL 12813 356  2 ng/mL 13610 715  5ng/mL 13465 730 10 ng/mL 13491 1395 20 ng/mL 12033 1718 50 ng/mL 118072696 100 ng/mL  12525 4131 200 ng/mL  11753 4876 500 ng/mL  9949 48761000 ng/mL  10797 5061

TABLE 5 0.2 μm sputum filtrate (second pass) readings spiked withvarying concentrations of BlaC Concentration CL Intensity TL IntensityNegative 10895 309 0.5 ng/mL  13388 426  1 ng/mL 13713 394  2 ng/mL14015 542  5 ng/mL 12332 689 10 ng/mL 12182 1344 20 ng/mL 11775 2122 50ng/mL 10297 2471 100 ng/mL  11989 4367 200 ng/mL  11991 4895 500 ng/mL 11755 5959 1000 ng/mL  11074 5456

Both 5 um (first pass) and 5 um/0.2 um (second pass) sputum filtratesshow similar performance and results for detection of BlaC in the sputumusing the test strips and Axxin reader. Sensitivity was as low as 2ng/mL of BlaC final concentration in each sample, demonstrating theutility of the antibody reagents and test apparatus.

IX. The following describes an assay demonstrating a successfuldetection of BlaC in a saliva sample using the lateral flow assay.

Saliva collection was performed by placing sponge (America Filtronasponge) in mouth for ten minutes to absorb saliva. Once saturated, thesponge was placed in a 5 mL syringe, and squeezed to collect saliva in a2 mL dolphin tube. The saliva was spiked with BlaC at a range ofconcentrations. The spiked saliva was mixed with running buffer (10 mMTris, 1% Tween-20, pH 7.2) at 1:1 ratio and vortexed to mix to providefinal concentrations of BlaC of 0.5 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL, 10ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL, 200 ng/mL, 500 ng/mL, and 1000ng/mL. 100 μL saliva-buffer mix was pipetted and run on strips(generated and assembled as described above). Results were read after 15minutes using an Axxin reader.

Results

The intensities of control line (CL) and test line (TL) signals asdetermined by the Axxin reader are set forth in Table 6 for the salivasamples. The TL intensities indicated in Table 6 are also graphicallyillustrated in FIG. 12.

TABLE 6 intensity readings for saliva spiked with varying concentrationsof BlaC Concentration CL Intensity TL Intensity Negative 7483 344 0.5ng/mL  8463 394  1 ng/mL 7729 635 2.5 ng/mL  7684 505  5 ng/mL 7601 571 7 ng/mL 7856 663 10 ng/mL 7326 626 12 ng/mL 7308 706 15 ng/mL 7729 72420 ng/mL 6918 826 25 ng/mL 7155 1167 30 ng/mL 6280 945 35 ng/mL 73381173 40 ng/mL 6812 1172 45 ng/mL 7866 1491 50 ng/mL 7229 1492 75 ng/mL7406 1628 100 ng/mL  6385 1572 200 ng/mL  8014 2199 500 ng/mL  6225 19431000 ng/mL  6524 2229

These results demonstrated that the disclosed anti-BlaC antibodies andnon-optimized lateral flow assay design can be used to successfullydetect BlaC antigen in saliva at a concentration at least as low as 0.5ng/mL.

X. The following describes ELISA assays demonstrating the use ofantibody reagent pairings that successfully detect recombinant andpurified wild-type BlaC, including additional assays to optimizeantibody concentrations and other reaction parameters. The assays alsodemonstrate lack of cross-reactivity to other antigens (i.e., have highspecificity for BlaC).

General ELISA Methods

Coating antibodies were diluted in sodium carbonate buffer (0.1 M SodiumCarbonate pH 9.5) to desired concentration and 100 uL were added to eachwell on a polycarbonate plate. The plate was incubated with theantibodies for 2 hours at 37° C. or overnight at 4° C. After incubation,the plate was washed 3× with PBS-Tw (1×PBS+0.1% Tween 20, pH 7.4).Antigen was diluted to the desired concentration with IX PBS (pH 7.4).100 uL of antigen was added to each well and incubated at 37° C. for 90minutes. After incubation, the plate was washed 3× with PBS-Tw. Theselected detection antibody diluted with PBS-Tw to desiredconcentration. 100 uL of the detection antibody was added to each welland incubated for 1 hour at 37° C. After incubation, the plates waswashed 3× with PBS-Tw. The HRP-conjugated antibody was diluted to thedesired titer with PBS-Tw. 100 uL was added to each well and incubatedfor 30 minutes at 37° C. After incubation, the plate was washed 2× withPBS-Tw, then another 2× with PBS. 100 uL of TMB substrate was added toeach well and the plate was incubated in the dark for 10-15 minutes. 50uL of IN sulfuric acid was added to stop the reaction. The plate wasread at 450 nm with 630 nm as the reference wavelength.

Results

1) ELISA Pairing Mouse Anti-BlaC mAb and Anti-BlaC Goat Polyclonal orAnti-BlaC Rabbit IgG

An initial screen was performed using 10 μg/mL Mouse anti-BlaC mAb (fromclone G1, as described above in part VI) as the coating (i.e., captureantibody) and 10 μg/mL anti-BlaC goat polyclonal (obtained by affinitypurification) or 10 μg/mL purified anti-BlaC rabbit IgG (polyclonal) asthe detection antibody. The bound detection antibody was monitored usingHRP-conjugated Ab at a titer of 1:8000 (rabbit) and 1:4000 (goat). It isnoted that the original concentration of HRP-rabbit and HRP-mouse aretwice that of HRP-goat, so titer for rabbit (or mouse) and goat isgenerally used at 2:1 ratio to adjust for this difference ofconcentration. BSA was used as a control. Recombinant BlaC (rBlaC) wasproduced as described above. Wild type BlaC was purified from 7H9cultures at 3 μg/mL (as the initial wtBlaC purification). “Sauton”refers to the supernatant from the growth medium in which M.tuberculosis was cultured, whereas Sauton (Broth (−)) is a negativecontrol medium with no M. tuberculosis cultured therein. Any positive orenhanced signal from the Sauton sample in contrast to the blank Sautonsample indicates the detection of wild-type BlaC excreted by the M.tuberculosis grown in the culture.

The results of the ELISA assays are set forth in Table 7.

TABLE 7 ELISA using Mouse anti-BlaC mAb (G1 clone) as capture antibodyand either anti-BlaC goat polyclonal or purified anti-BlaC rabbit IgG asdetection antibody for various antigens Detection: Detection: AntigenGoat pAb Rabbit pAb 0.1% BSA 0.883 0.139 0.1% BSA 0.679 0.139 rBlaC (100ng/mL) 1.43 1.493 rBlaC (500 ng/mL) 1.951 1.514 wt BlaC (1:10 dilut)1.263 0.677 wt BlaC (1:100 dilut) 1.382 0.781 Sauton (OD: 1.0) 1.5481.116 Sauton (Broth (−)) 0.926 0.151

These results demonstrate that the combination of Mouse anti-BlaC mAb(G1 clone) and either anti-BlaC goat polyclonal or anti-BlaC rabbit IgGantibodies is useful to detect both recombinant and wild-type BlaC.Furthermore, BlaC is detectable from the supernatant of M. tuberculosis,further indicating the utility of these reagents for the detection ofTB-complex bacteria from a sample.

2) ELISA Pairing Anti-BlaC Rabbit IgG with Three Mouse Anti-BlaC mAbs

An expanded screen was performed using 10 μg/mL and 20 μg/mL of Mouseanti-BlaC mAb (from clone G1, H1, and 31A, as described above in partVI) as the coating (“C” i.e., capture antibody) and 10 μg/mL purifiedanti-BlaC rabbit polyclonal IgG Ab as the detection antibody (“D”). Thebound detection antibody was monitored using HRP-conjugated Ab at atiter of 1:8000 (rabbit). BSA and Sauton (−) were used as controls.rBlaC, wtBlaC, and Sauton (BlaC+), as described above in part (1), wereused as antigen.

The results of the ELISA assays are set forth in Table 8.

TABLE 8 ELISA pairing anti-BlaC rabbit IgG (D) with three Mouseanti-BlaC mAbs (C) D: 10 ug/mL D: 10 ug/mL D: 10 ug/mL D: 10 ug/mL D: 10ug/mL D: 10 ug/mL C: G1 C: H1 C: 31A C: G1 C: H1 C: 31A 10 ug/mL 10ug/mL 10 ug/mL 20 ug/mL 20 ug/mL 20 ug/mL 0.1% BSA 0.905 1.139 0.9120.946 1.26 0.902 0.1% BSA 0.849 0.93 0.774 0.893 1.131 0.863rBlaC, >3.0 >3.0 >3.0 >3.0 >3.0 >3.0 20 ng/mLrBlaC, >3.0 >3.0 >3.0 >3.0 >3.0 >3.0 50 ng/mL wtBlaC, 1:10 2.48 2.9382.214 2.574 >3.0 2.075 dilut wtBlaC, 1:100 2.434 >3.0 2.213 2.681 2.9732.161 dilut Sauton (+), >3.0 >3.0 >3.0 >3.0 >3.0 >3.0 OD 1.0 Sauton (−),1.065 1.225 1.008 1.029 1.576 1.281 Broth

These results indicate that all three Mouse monoclonal antibodies (i.e.,from clones G1, H1, and 31A) were effective in pairing with anti-BlaCrabbit IgG as a detection antibody for specifically detectingrecombinant and wildtype BlaC as compared to BSA control. Furthermore,these reagents were effective in detecting BlaC in the growth mediumfrom M. tuberculosis culture.

3) Antibody Titer Assay

An antibody titer assay was performed using varying amounts (10 μg/mL,g/mL, 2.5 μg/mL, and 1.25 μg/mL) of Mouse anti-BlaC mAb (from clone G1,as described above in part VI) as the coating (“C” i.e., captureantibody) and varying amounts (10 μg/mL, 5 μg/mL, and 2.5 μg/mL) ofpurified anti-BlaC rabbit IgG as the detection antibody (“D”). The bounddetection antibody was monitored using HRP-conjugated Ab at a titer of1:10,000 (rabbit). BSA and Sauton (−) were used as controls. rBlaC,wtBlaC, and Sauton (BlaC+), as described above in part (1), were used asantigen.

The results of the ELISA assays are set forth in Table 9.

TABLE 9 ELISA titer assay using varying amounts of Mouse anti-BlaC mAb(C) and anti- BlaC rabbit IgG (D) wtBlaC, Sauton 0.1% 0.1% rBlaC, rBlaC,wtBlaC, 1:100 (+), OD Sauton BSA BSA 20 ng/mL 50 ng/mL 1:10 dilut dilut1.0 (−), Broth D: 10 ug/m 0.728 0.794 >3.0 >3.0 2.263 2.256 >3.0 0.978C: 10 ug/mL D: 5 ug/mL 0.498 0.502 >3.0 >3.0 1.556 1.616 >3.0 0.605 C:10 ug/mL D: 2.5 ug/mL 0.313 0.319 >3.0 >3.0 1.022 1.008 >3.0 0.361 C: 10ug/mL D: 10 ug/mL 0.755 0.679 >3.0 >3.0 2.138 2.189 >3.0 0.922 C: 5ug/mL D: 5 ug/mL 0.548 0.412 >3.0 >3.0 1.398 1.573 >3.0 0.597 C: 5 ug/mLD: 2.5 ug/mL 0.305 0.268 >3.0 >3.0 0.968 0.959 >3.0 0.394 C: 5 ug/mL D:10 ug/mL 0.763 0.654 >3.0 >3.0 2.189 2.249 >3.0 0.809 C: 2.5 ug/mL D: 5ug/mL 0.443 0.395 >3.0 >3.0 1.33 1.212 >3.0 0.57 C: 2.5 ug/mL D: 2.5ug/mL 0.243 0.265 >3.0 >3.0 0.85 0.798 2.854 0.322 C: 2.5 ug/mL D: 10ug/mL 0.582 0.56 >3.0 >3.0 1.368 1.691 >3.0 0.727 C: 1.25 ug/mL D: 5ug/mL 0.341 0.294 >3.0 >3.0 0.795 0.812 >3.0 0.471 C: 1.25 ug/mL D: 2.5ug/mL 0.182 0.178 2.877 >3.0 0.449 0.468 2.189 0.272 C: 1.25 ug/mL

These results demonstrate that even at the lowest tested antibodyconcentrations for both the capture (coating) and detection antibodies(see column 12), the ELISA assay was able to specifically detectrecombinant and wild-type BlaC.

4) Antibody Titer Assay, Continued

A further antibody titer assay was performed using varying amounts (0.6μg/mL, 0.3 μg/mL, and 0.15 μg/mL) of Mouse anti-BlaC mAb (from clonesG1, H1, and 31A, as described above in part VI) as the coating (“C”i.e., capture antibody) and 2.5 μg/mL of purified anti-BlaC rabbit IgGpolyclonal Ab as the detection antibody (“D”). The bound detectionantibody was monitored using HRP-conjugated Ab at a titer of 1:12,000(rabbit). PBS, BSA and Sauton (−) were used as controls. rBlaC, andSauton (BlaC+), as described above in part (1), were used as antigen.

The results of the ELISA assays are set forth in Table 10.

TABLE 10 ELISA titer assay using varying amounts of Mouse anti-BlaC mAb(C) and 2.5 μg/mL of purified anti-BlaC rabbit IgG polyclonal Ab as thedetection antibody (D) 0.1% 0.1% rBlaC rBlaC Sauton (+), Sauton (−),PBS-T PBS-T BSA BSA 2 ng/mL 5 ng/mL OD 1.0 Broth C: G1 0.128 0.148 0.1040.098 0.208 0.23 1.504 0.155 0.6 ug/mL C: H1 0.159 0.164 0.134 0.1070.288 0.496 1.53 0.156 0.6 ug/mL C: 31A 0.161 0.204 0.104 0.092 0.2660.363 1.461 0.16 0.6 ug/mL C: G1 0.106 0.108 0.088 0.088 0.164 0.2171.843 0.12 0.3 ug/mL C: H1 0.135 0.124 0.11 0.097 0.165 0.261 1.68 0.1250.3 ug/mL C: 31A 0.121 0.12 0.085 0.084 0.18 0.248 1.657 0.147 0.3 ug/mLC: G1 0.098 0.105 0.075 0.071 0.134 0.196 1.526 0.089 0.15 ug/mL C: H10.095 0.1 0.093 0.073 0.171 0.214 1.484 0.203 0.15 ug/mL C: 31A 0.0960.095 0.067 0.068 0.161 0.209 1.527 0.12 0.15 ug/mL

These results demonstrate that even at the lowest tested antibodyconcentrations for both the capture (coating) and detection antibodies(see column 12), the ELISA assay was able to specifically detect BlaCover blank and irrelevant protein controls.

5) Antibody Titer Assay, Continued

A further antibody titer assay was performed using varying amounts (0.6μg/mL and 0.3 μg/mL) of Mouse anti-BlaC mAb (from clones G1, H1, and31A, as described above in part VI) as the coating (“C” i.e., captureantibody) and 2.5 μg/mL of purified anti-BlaC Goat or rabbit anti-BlaCserum (polyclonal serum at 8 week post-immunization) as the detectionantibody (“D”). The bound detection antibody was monitored usingHRP-conjugated Ab at a titer of 1:10,000 (rabbit) or 1:5,000 (goat).PBS, BSA and Sauton (−) were used as controls. rBlaC, wtBlaC, and Sauton(BlaC+), as described above in part (1), were used as antigen.

The results of the ELISA assays are set forth in Table 11.

TABLE 11 ELISA titer assay using varying amounts of Mouse anti-BlaC mAbfrom clones G1, H1, and 31A (C) and 2.5 ug/mL of purified anti-BlaC Goator rabbit anti-BlaC serum as the detection antibody (D) 0.1% 0.1% rBlaCrBlaC Sauton (+), Sauton (−), PBS-T PBS-T BSA BSA 2 ng/mL 5 ng/mL OD 1.0Broth D: Goat; 2.416 2.42 1.513 1.779 2.413 2.444 2.179 2.201 C: G1 (0.6μg/mL) D: Goat; >3.0 >3.0 2.374 2.403 >3.0 >3.0 >3.0 >3.0 C: H1 (0.6μg/mL) D: Goat; 2.009 2.019 1.351 1.275 1.936 1.909 1.868 1.837 C: 31A(0.6 μg/mL) D: Goat; 1.453 1.352 0.941 0.892 1.292 1.403 1.432 1.247 C:G1 (0.3 μg/mL) D: Goat; 1.634 1.892 1.111 1.161 1.648 1.814 1.661 1.674C: H1 (0.3 μg/mL) D: Goat; 1.05 1.01 0.701 0.742 1.058 1.238 1.136 1.022C: 31A (0.3 μg/mL) D: Rab; 0.375 0.362 0.16 0.199 0.402 0.457 0.6260.357 C: G1 (0.6 μg/mL) D: Rab; 0.397 0.397 0.209 0.188 0.438 0.6471.441 0.402 C: H1 (0.6 μg/mL) D: Rab; 0.34 0.309 0.215 0.214 0.411 0.4490.629 0.37 C: 31A (0.6 μg/mL) D: Rab; 0.358 0.364 0.155 0.165 0.4020.409 0.553 0.401 C: G1 (0.3 μg/mL) D: Rab; 0.461 0.556 0.252 0.2240.796 0.925 0.627 0.652 C: H1 (0.3 μg/mL) D: Rab; 0.216 0.339 0.1810.151 0.378 0.343 0.544 0.269 C: 31A (0.3 μg/mL)

These results demonstrate that some combinations of the capture anddetection antibodies (i.e., anti-BlaC Goat or rabbit anti-BlaC serum),at the lowest tested antibody concentrations retained the ability tospecifically detect BlaC over blank and irrelevant protein controls.However, other combinations were unable to reliably provide a noticeabledetectable signal improvement over the controls.

6) ELISA Assay Using Mouse Anti-BlaC mAb and Low HRP Titer

An ELISA assay was performed using varying amounts (0.15 μg/mL and 0.075μg/mL) of Mouse anti-BlaC mAb (from clones G1, H1, and 31A, as describedabove in part VI) as the coating (“C” i.e., capture antibody) and 2.5μg/mL of purified anti-BlaC rabbit IgG polyclonal Ab as the detectionantibody (“D”). The bound detection antibody was monitored usingHRP-conjugated Ab at a titer of 1:9,000 (rabbit). PBS, BSA and Sauton(−) were used as controls. rBlaC, wtBlaC, and Sauton (BlaC+), asdescribed above in part (1), were used as antigen.

The results of the ELISA assays are set forth in Table 12.

TABLE 12 ELISA titer assay using varying amounts of Mouse anti-BlaC mAbfrom clones G1, H1, and 31A (C) and 2.5 μg/mL of purified anti-BlaCrabbit IgG as the detection antibody (D) C: G1 C: H1 C: 31A C: G1 C: H1C: 31A Antigen 0.15 ug/mL 0.15 μg/mL 0.15 μg/mL 0.075 μg/mL 0.075 μg/mL0.075 μg/mL PBS-T 0.327 0.176 0.18 0.176 0.22 0.166 PBS-T 0.183 0.1570.186 0.176 0.318 0.204 0.1% BSA 0.141 0.13 0.125 0.144 0.239 0.156 0.1%BSA 0.141 0.124 0.132 0.189 0.162 0.186 rBlaC 0.396 .0449 0.385 0.1930.436 0.387 5 ng/mL rBlaC 0.296 0.244 0.214 0.391 0.241 0.21 2 ng/mLrBlaC 0.195 0.231 0.206 0.245 0.321 0.18 1 ng/mL Sauton (+), 2.561 2.7372.743 0.204 2.916 2.561 OD 0.8 Sauton (+), 0.983 1.243 1.122 2.741 1.1381.096 OD 0.5 or ~10⁷ CFU/ml Sauton (+), 0.242 0.194 0.199 1.298 0.3060.151 10⁵ CFU/ml Sauton (+), 0.156 0.172 0.152 0.159 0.196 0.138 10³CPU/ml Sauton (−), 0.197 0.168 0.169 0.174 0.158 0.15 Broth

As illustrated in Table 12, the decreasing levels of BlaC in the Sautonantigen were still detectable using all of the antibody combinationsover the blank Sauton antigen control until the antigen-positive Sautonmixture reached 103 CFU/ml. Furthermore, these results generallydemonstrate that most combinations of the capture and detectionantibodies, even at the lowest tested concentrations, retained theability to specifically detect BlaC over blank and irrelevant proteincontrols. However, other combinations were unable to reliably provide anoticeable detectable signal improvement over the controls.

7) ELISA Assay Using Mouse Anti-BlaC mAb Paired with and 2.5 μg/mL ofPurified Anti-BlaC Rabbit IgG

An ELISA assay was performed using varying amounts (0.15 μg/mL and 0.075μg/mL) of Mouse anti-BlaC mAb (from clones G1, H1, and 31A, as describedabove in part VI) as the coating (“C” i.e., capture antibody) and 2.5μg/mL of purified anti-BlaC rabbit IgG polyclonal Ab as the detectionantibody (“D”). The bound detection antibody was monitored usingHRP-conjugated Ab at a titer of 1:12,000 (rabbit). PBS, BSA and Sauton(−) were used as controls. rBlaC, wtBlaC, and Sauton (BlaC+), asdescribed above in part (1), were used as antigen.

The results of the ELISA assays are set forth in Table 13.

TABLE 13 ELISA titer assay using varying amounts of Mouse anti-BlaC mAbfrom clones G1, H1, and 31A (C) and 2.5 ug/mL of purified anti-BlaCrabbit IgG as the detection antibody (D) C: G1 C: H1 C: 31A C: G1 C: H1C: 31A Antigen 0.15 ug/mL 0.15 μg/mL 0.15 μg/mL 0.075 μg/mL 0.075 μg/mL0.075 μg/mL PBS-T 0.115 0.152 0.15 0.16 0.152 0.209 PBS-T 0.132 0.1510.129 0.15 0.165 0.164 0.1% BSA 0.086 0.109 0.118 0.113 0.122 0.466 0.1%BSA 0.085 0.126 0.085 0.13 0.164 0.152 rBlaC 0.275 0.285 0.275 0.2470.424 0.279 5 ng/mL rBlaC 0.216 0.198. 0.173 0.181 0.193 0.182 2 ng/mLrBlaC 0.164 0.17 0.154 0.157 0.15 0.145 1 ng/mL Sauton (+), 1.738 1.5612.003 2.095 1.806 2.801 OD 0.8 Sauton (+), 0.761 0.692 0.709 0.922 0.8740.984 OD 0.5 or ~10⁷ CFU/ml Sauton (+), 0.131 0.2 0.162 0.124 0.2840.143 10⁵ CFU/ml Sauton (+), 0.135 0.131 0.156 0.127 0.247 0.121 10³CFU/ml Sauton (−), 0.154 0.151 0.161 0.149 0.133 0.14 Broth

As illustrated in Table 13, the decreasing levels of BlaC in the Sautonantigen were still detectable using all of the antibody combinationsover the blank Sauton antigen control until the antigen-positive Sautonmixture reached 10³ CFU/ml. However, it is noted that the BlaC antigenwas still detectable in the most dilute BlaC-positive Sauton antigenusing the H1 Mouse mAb as the capture reagent. Furthermore, theseresults demonstrate that some combinations of the capture and detectionantibodies, even at the lowest tested antibody concentrations, retainedthe ability to specifically detect BlaC over blank and irrelevantprotein controls. However, other combinations were unable to reliablyprovide a noticeable detectable signal improvement over the controls.

Furthermore, FIG. 13 graphically illustrates the signal intensity forthe Mouse anti-BlaC H1 mAb in detecting the BlaC present in the negativecontrols as compared to the BlaC positive Sauton antigen samples. Asindicated in the graph, the combination of the H1 mAb with the purifiedanti-BlaC rabbit (polyclonal) IgG detection antibody provided a gooddifference between negative control and the minimal presence (i.e., 10³CFU) of M. tuberculosis in the Sauton's culture medium. This contributesto the other assay results, which in combination indicate that at least11 different pairs from three monoclonal antibodies and four polyclonalantibodies can provide sensitive detection of wild-type and recombinantBlaC.

8) ELISA Assay Using Mouse Anti-BlaC Paired with and 1.0 μg/mL ofAffinity Purified Anti-BlaC Goat Polyclonal Ab

An ELISA assay was performed using varying amounts (0.15 μg/mL and 0.075μg/mL) of Mouse anti-BlaC mAb (from clones G1, H1, and 31A, as describedabove in part VI) as the coating (“C” i.e., capture antibody) and 1.0μg/mL of Goat affinity purified anti-BlaC (affinity purified) polyclonalantibody as the detection antibody (“D”). The bound detection antibodywas monitored using HRP-conjugated Ab at a titer of 1:12,000 (rabbit).PBS, BSA and Sauton (−) were used as controls. rBlaC, wtBlaC, and Sauton(BlaC+), as described above in part (1), were used as antigen.

The results of the ELISA assays are set forth in Table 14.

TABLE 14 ELISA titer assay using varying amounts of Mouse anti-BlaC mAbfrom clones G1, H1, and 31A (C) and 1.0 ug/mL of affinity purifiedanti-BlaC Goat polyclonal as the detection antibody (D) C: G1 C: H1 C:31A C: G1 C: H1 C: 31A Antigen 0.15 ug/mL 0.15 μg/mL 0.15 μg/mL 0.075μg/mL 0.075 μg/mL 0.075 μg/mL PBS-T 0.42 0.461 0.332 0.227 0.237 0.249PBS-T 0.446 0.482 0.349 0.262 0.224 0.203 0.1% BSA 0.34 0.33 0.24 0.160.167 0.146 0.1% BSA 0.333 0.33 0.258 0.159 0.182 0.139 rBlaC 0.587 0.650.53 0.91 0.481 0.411 1 ng/mL rBlaC 0.73 0.869 0.798 0.644 0.683 0.609 2ng/mL rBlaC 0.137 1.341 1.307 1.198 1.222 1.235 5 ng/mL Sauton (+),1.164 1.636 1.132 1.174 1.088 1.559 OD 0.8 Sauton (+), 0.71 0.718 0.770.766 0.598 0.628 OD 0.5 or ~10⁷ CFU/ml Sauton (+), 0.586 0.384 0.3110.21 0.224 0.18 10⁵ CFU/ml Sauton (+), 0.365 0.405 0.1 0.204 0.215 0.35610³ CFU/ml Sauton (−), 0.408 0.414 0.295 0.195 0.196 0.17 Broth

As illustrated in Table 14, the decreasing levels of BlaC in the Sautonmedium were still detectable using all of the antibody combinations(with the capture antibody at 0.075 μg/mL) over the blank Sauton antigencontrol. Interestingly, when the capture antibodies were coated at thehigher concentration, the lowest Sauton (+) signal was indistinguishablefrom the blank Sauton medium. Furthermore, all combinations of thecapture and detection antibodies, even at the lowest testedconcentrations, retained the ability to specifically detect the lowestlevels of rBlaC over blank and irrelevant protein controls.

9) ELISA Assay Using Mouse Anti-BlaC mAb as Detection Antibodies Pairedwith Various Polyclonal Antibodies as Capture Reagent

An ELISA assay was performed using 0.15 μg/mL various anti-BlaCpolyclonal antibodies (i.e., affinity purified goat, purified goat IgG,rabbit serum (“A&M”; 8-week post immunization bleed), and rabbit Abcam)as the coating (“C” i.e., capture antibody) and 2.5 μg/mL of Mouseanti-BlaC mAb (from clones G1, H1, and 31A, as described above in partVI) as the detection antibody (“D”). The bound detection antibody wasmonitored using HRP-conjugated Ab at a titer of 1:12,000 (mouse). PBS,BSA and Sauton (−) were used as controls. rBlaC, wtBlaC, and Sauton(BlaC+), as described above in part (1), were used as antigen.

The results of the ELISA assays are set forth in Table 15.

TABLE 15 ELISA titer assay using 2.5 ug/mL of Mouse anti-BlaC mAb fromclones G1, H1, and 31A (D) and 0.15 ug/mL of various anti-BlaCpolyclonal reagents as the detection antibody (C) Sauton 0.1% 0.1% rBlaCrBlaC (+), OD Sauton PBS-T PBS-T BSA BSA 5 ng/mL 2 ng/mL 0.8 (−), BrothD: Goat Aff 0.074 0.086 1.627 1.78 1.912 0.879 0.634 0.071 C: G1 D: GoatIgG 0.106 0.092 1.768 1.85 1.85 0.953 0.609 0.136 C: G1 D: A&M rab 0.0870.089 1.617 1.661 1.793 0.885 0.646 0.077 C: G1 D: Abcam rab 0.09 0.0811.668 1.851 2.03 0.939 0.604 0.076 C: G1 D: Goat Aff 0.1074 0.12 0.3990.422 1.599 0.712 0.599 0.172 C: H1 D: Goat IgG 0.138 0.103 0.364 0.3841.426 0.64 0.399 0.114 C: H1 D: A&M rab 0.1 0.103 0.352 0.381 1.426 0.670.373 0.109 C: H1 D: Abcam rab 0.11 0.099 0.366 0.36 1.523 0.729 0.4410.111 C: H1 D: Goat Aff 0.17 0.17 >3.0 >3.0 0.68 0.402 0.387 0.181 C:31A D: Goat IgG 0.139 0.135 >3.0 >3.0 0.521 0.353 0.395 0.149 C: 31A D:A&M rab 0.133 0.135 >3.0 >3.0 0.482 0.315 0.405 0.127 C: 31A D: Abcamrab 0.144 0.147 >3.0 >3.0 0.553 0.331 0.495 0.144 C: 31A

As illustrated in Table 15, all assays incorporating a Mouse anti-BlaCmonoclonal antibody as a detection antibody in combination with apolyclonal reagent as the capture antibody provided significantly higherBlaC signal in the BlaC (+) Sauton's medium and BlaC antigen groups overthe blank Sauton medium and PBS control. This demonstrates, inconnection with the above assays, that the monoclonal antibodies areuseful in a variety of formats including when used as immobilizedcapture reagents or as detection reagents that can flow across a strip.

10) ELISA Cross-Reactivity Assay Comparing ELISA Detection of BlaC andNon TB-Complex β-Lactamases

To test the cross reactivity of an assay using effective detection andcapture reagents, the best concentration conditions from part (7),above, were used. Mouse anti-BlaC monoclonal antibodies from clones G1,H1, and 31A were used as the coat (i.e., “capture” (C)) reagent inranges from 0.15 to 0.075 μg/mL. Purified anti-IgG rabbit polyclonalreagent was used as the detection antibody (“D”) at 2.5 μg/mL. Any bounddetection antibody was quantified using HRP-conjugated Ab at a titer of1:12,000 (rabbit). Assays were run for TEM-1, a non TB-complexβ-lactamase (Texas A&M University) produced from E. coli, a nonTB-complex β-lactamase Type II (Sigma), and a non TB-complex β-lactamaseType IV from Enterobacter cloacae (Sigma). Specifically, the three nonTB-complex β-lactamases were each applied at concentrations of 1 μg/mL,500 ng/mL, 200 ng/mL, 100 ng/mL, 50 ng/mL, 20 ng/mL 10 ng/mL, and 5ng/mL. Tests were conducted in PBS using the non-optimized ELISA formatsotherwise described above.

The results were compared with the prior results obtained from M.tuberculosis (i.e., TB complex BlaC) in equivalent ELISA conditions. Nocross reactivity of the assay (incorporating Mouse anti-BlaC mAbs andpurified rabbit IgG anti-Blac polyclonal reagent) was observed for anyof the non TB-complex β-lactamase. FIG. 14 graphically illustrates arepresentative comparison of signal observed for the differentβ-lactamase antigens (or PBS control) when applied at 5 ng/mL. TheTB-complex BlaC provided a signal about four times background signal,whereas the non TB-complex β-lactamase did not differ from background.Comparison to the non TB-complex β-lactamases was performed to ascertainlikelihood of any false positive signals and, thus, the utility of a TBdiagnostic assay based on the disclosed reagents. However, it isuncertain which, or how much, if any of the β-lactamases from non TBcomplex bacteria might ever be present in the sputum or other biologicalsample obtained from a subject suspected of an infection. Accordingly,to demonstrate the amounts of the tested non TB-complex β-lactamasesneeded to equate to a 5 ng signal of BlaC, the maximum amount of the nonTB-complex β-lactamases that produced an equivalent signal with theindicated ELISA format are represented graphically in FIG. 15. It isnoted that the signal for β-lactamase Type II represents the maximumamount tested and yet still did not produce an “equivalent” signal,re-affirming a complete lack of functional cross-reactivity.

11) ELISA Optimization Assay: Mouse Anti-BlaC mAb (G1) and Amount ofrBlaC Antigen

An optimization ELISA was performed using from 2 μg/mL to 0.075 μg/mLMouse anti-BlaC mAb (from clone G1, as described above in part VI) asthe coating (“C” i.e., capture antibody) and 2.5 μg/mL purified rabbitanti-BlaC polyclonal Ab as the detection antibody (“D”). The bounddetection antibody was monitored using HRP-conjugated Ab at a titer of1:12,000 (rabbit). PBS and 0.25 ng/mL to 20 ng/mL recombinant BlaC,generated as described above, were used as antigen. Each antigencondition was run in duplicate.

The results of the ELISA optimization assay are set forth in Table 16.

TABLE 16 ELISA assessing titers of Mouse anti-BlaC mAb from clone G1 (C)paired with 2.5 μg/mL purified rabbit anti-BlaC polyclonal antibody asthe detection antibody (D) with indicated amounts of rBlaC antigen. C:0.3 C: 0.15 C: .075 C: 2 μg C: 1 μg C: 0.6 μg μg μg μg PBS 0.44 0.4360.289 0.333 0.275 0.217 PBS 0.432 0.462 0.343 0.404 0.287 0.245 rBlaC0.473 0.386 0.354 0.319 0.282 0.252 0.25 ng/mL rBlaC 0.435 0.417 0.4350.33 0.306 0.28 0.25 ng/mL rBlaC 0.521 0.467 0.42 0.337 0.325 0.278 0.5ng/mL rBlaC 0.56 0.492 0.402 0.322 0.309 0.27 0.5 ng/mL rBlaC 0.7220.514 0.406 0.34 0.359 0.282 1 ng/mL rBlaC 0.684 0.571 0.44 0.398 0.3530.709 1 ng/mL rBlaC 0.959 0.872 0.617 0.464 0.541 0.408 2.5 ng/mL rBlaC0.914 0.775 0.588 0.464 0.438 0.359 2.5 ng/mL rBlaC 1.554 1.339 0.9620.742 0.709 0.564 5 ng/mL rBlaC 1.532 0.481 0.903 0.719 0.666 0.593 5ng/mL rBlaC >3 2.638 1.425 0.734 0.466 0.385 10 ng/mL rBlaC >3 2.5651.14 0.801 0.471 0.395 10 ng/mL rBlaC >3 >3 2.941 2.451 2.434 2.099 20ng/mL rBlaC >3 >3 >3 2.48 2.307 1.855 20 ng/mL

Table 16 illustrates the signal resulting from varying levels of captureantibody (Mouse anti-BlaC mAb from clone G1) and varying amounts ofantigen.

12) ELISA Optimization Assay: Mouse Anti-BlaC mAb (H1) and Amount ofrBlaC Antigen

An optimization ELISA was performed using from 2 μg/mL to 0.075 μg/mLMouse anti-BlaC mAb (from clone G1, as described above in part VI) asthe coating (“C” i.e., capture antibody) and 2.5 μg/mL purified rabbitanti-BlaC polyclonal antibody as the detection antibody (“D”). The bounddetection antibody was monitored using HRP-conjugated Ab at a titer of1:12,000 (rabbit). PBS and 0.25 ng/mL to 20 ng/mL recombinant BlaC,generated as described above, were used as antigen. Each antigencondition was run in duplicate.

The results of the ELISA optimization assay are set forth in Table 17.

TABLE 17 ELISA assessing titers of Mouse anti-BlaC mAb from clone H1 asthe capture antibody (C) paired with 2.5 μg/mL purified rabbit anti-BlaCpolyclonal antibody as the detection antibody (D) with indicated amountsof rBlaC antigen. C: 0.3 C: 0.15 C: .075 C: 2 μg C: 1 μg C: 0.6 μg μg μgμg PBS 0.598 0.56 0.456 0.36 0.261 0.244 PBS 0.547 0.516 0.421 0.3080.295 0.85 rBlaC 0.612 0.539 0.422 0.321 0.329 0.24 0.25 ng/mL rBlaC0.645 0.615 0.446 0.49 0.356 0.228 0.25 ng/mL rBlaC 0.639 0.595 0.4390.315 0.308 0.288 0.5 ng/mL rBlaC 0.709 0.601 0.415 0.503 0.312 0.2930.5 ng/mL rBlaC 0.738 0.696 0.495 0.335 0.257 0.27 1 ng/mL rBlaC 0.7040.736 0.505 0.328 0.327 0.282 1 ng/mL rBlaC 1.072 1.056 0.774 0.5480.405 0.385 2.5 ng/mL rBlaC 1.08 1.095 0.757 0.463 0.433 0.394 2.5 ng/mLrBlaC 1.674 1.668 1.177 0.707 0.646 0.592 5 ng/mL rBlaC 1.789 1.6881.177 0.705 0.629 0.553 5 ng/mL rBlaC >3 >3 2.311 0.893 0.554 0.37 10ng/mL rBlaC >3 >3 2.487 0.752 0.495 0.375 10 ng/mL rBlaC >3 >3 >3 2.4282.06 1.715 20 ng/mL rBlaC >3 >3 >3 2.606 1.928 1.526 20 ng/mL

Table 17 illustrates the signal resulting from varying levels of captureantibody (Mouse anti-BlaC mAb from clone H1) and varying amounts ofantigen.

13) ELISA Optimization Assay: TCEP

An optimization ELISA was performed where the antigen solution wassubject to the treatment with varying amounts of TCEP(tris(2-carboxyethyl)phosphine), which is a reducing agent that breaksdisulfide bond, to determine the effect of such reduction/denaturizationon the ability for the Mouse anti-BlaC mAbs and partner reagents todetect BlaC. Between 20 mM and 40 mM TCEP, at varying ng/mL, weretested. The ELISAs used 0.15 μg/mL and 0.075 μg/mL of Mouse anti-BlaCmAb (from clones G1, H1, and 31A, as described above in part VI) as thecoating (“C” i.e., capture antibody) and 2.5 μg/mL purified rabbitanti-BlaC polyclonal antibody as the detection antibody (“D”). The bounddetection antibody was monitored using HRP-conjugated Ab at a titer of1:12,000 (rabbit). PBS and recombinant BlaC, generated as describedabove, were used as antigen.

The results of the ELISA optimization assay are set forth in Table 18.

TABLE 18 ELISA assessing titers of TCEP on the ability of Mouseanti-BlaC mAb from clones G1, H1, and 31A as the capture antibody (C),paired with 2.5 μg/mL purified rabbit anti-BlaC polyclonal antibody asthe detection antibody (D), to detect BlaC antigen. C: G1 C: H1 C: 31AC: G1 C: H1 C: 31A 0.15 μg/mL 0.15 μg/mL 0.15 μg/mL .075 μg/mL .075μg/mL .075 μg/mL 20 mM TCEP 0.048 0.048 0.052 0.054 0.059 0.054 20 mMTCEP 0.088 0.089 0.065 0.074 0.077 0.082 5 ng/mL 20 mM TCEP 0.082 0.1090.083 0.106 0.091 0.093 10 ng/mL 20 mM TCEP 0.102 0.116 0.133 0.1240.123 0.121 20 ng/mL 30 mM TCEP 0.093 0.067 0.049 0.07 0.057 0.074 30 mMTCEP 0.06 0.065 0.084 0.075 0.096 0.067 5 ng/mL 30 mM TCEP 0.068 0.090.087 0.094 0.083 0.094 10 ng/mL 30 mM TCEP 0.084 0.083 0.094 0.12 0.10.133 20 ng/mL 40 mM TCEP 0.044 0.068 0.049 0.062 0.058 0.079 40 mM TCEP0.056 0.06 0.058 0.071 0.07 0.062 5 ng/mL 40 mM TCEP 0.063 0.07 0.0630.076 0.065 0.073 10 ng/mL 40 mM TCEP 0.214 0.093 0.072 0.083 0.0750.122 20 ng/mL

Table 18 illustrates the effect of varying amounts of TCEP on theability of the Mouse anti-BlaC mAbs to pair with purified rabbitanti-BlaC polyclonal antibody to detect BlaC.

14) ELISA Optimization Assay: DTT

An optimization ELISA was performed where the antigen solution wassubject to the treatment with varying amounts of DTT (dithiothreitol),which is a strong reducing agent that breaks disulfide bond, todetermine the effect of such reduction/denaturization on the ability forthe Mouse anti-BlaC mAbs and partner reagents to detect BlaC. Between0.05% and 0.2% DTT, at varying ng/mL, were tested. The ELISAs used 0.15μg/mL and 0.075 μg/mL of Mouse anti-BlaC mAb (from clones G1, H1, and31A, as described above in part VI) as the coating (“C” i.e., captureantibody) and 2.5 μg/mL purified rabbit anti-BlaC polyclonal antibody asthe detection antibody (“D”). The bound detection antibody was monitoredusing HRP-conjugated Ab at a titer of 1:12,000 (rabbit). PBS andrecombinant BlaC, generated as described above, were used as antigen.

The results of the ELISA optimization assay are set forth in Table 19.

TABLE 19 ELISA assessing titers of DTT on the ability of Mouse anti-BlaCmAb from clones G1, H1, and 31A as the capture antibody (C), paired with2.5 μg/mL purified rabbit anti-BlaC polyclonal antibody as the detectionantibody (D), to detect BlaC antigen. C: G1 C: H1 C: 31A C: G1 C: H1 C:31A 0.15 μg/mL 0.15 μg/mL 0.15 μg/mL .075 μg/mL .075 μg/mL .075 μg/mL0.05% DTT 0.501 0.086 0.06 0.072 0.058 0.063 0.05% DTT 0.066 0.076 0.1680.074 0.076 0.064 5 ng/mL 0.05% DTT 0.062 0.081 0.072 0.098 0.074 0.06510 ng/mL 0.05% DTT 0.091 0.068 0.084 0.08 0.069 0.07 20 ng/mL 0.1% DTT0.052 0.057 0.064 0.062 0.061 0.056 0.1% DTT 0.051 0.072 0.058 0.0690.065 0.059 5 ng/mL 0.1% DTT 0.054 0.072 0.066 0.065 0.058 0.058 10ng/mL 1% DTT 0.067 0.074 0.07 0.103 0.075 0.072 20 ng/mL 0.2% DTT 0.0470.059 0.058 0.064 0.069 0.057 0.2% DTT 0.052 0.06 0.055 0.066 0.0620.056 5 ng/mL 0.2% DTT 0.054 0.066 0.076 0.062 0.727 0.194 10 ng/mL 0.2%DTT 0.06 0.067 0.072 0.1 0.066 0.074 20 ng/mL

Table 19 illustrates the effect of varying amounts of DTT on the abilityof the Mouse anti-BlaC mAbs to pair with purified rabbit anti-BlaCpolyclonal antibody to detect BlaC.

15) ELISA Optimization Assay: Purification of BlaC by Q Column

An optimization ELISA was performed where various amounts of BlaCantigen purified by Q column, which is based on ion exchange, were usedto determine the effect of such isolation on the ability for the Mouseanti-BlaC mAbs and partner reagents to detect the rBlaC. The ELISAs used0.15 μg/mL and 0.075 μg/mL of Mouse anti-BlaC mAb (from clones G1, H1,and 31A, as described above in part VI) as the coating (“C” i.e.,capture antibody) and 2.5 μg/mL purified rabbit anti-BlaC polyclonalantibody as the detection antibody (“D”). The bound detection antibodywas monitored using HRP-conjugated Ab at a titer of 1:12,000 (rabbit).PBS and between 10 ng/mL and 500 ng/mL Q column-purified rBlaC were usedas antigen.

The results of the ELISA optimization assay are set forth in Table 20.

TABLE 20 ELISA Q column antigen purification on the ability of Mouseanti-BlaC mAb from clones G1, H1, and 31A as the capture antibody (C),paired with 2.5 μg/mL purified rabbit anti-BlaC polyclonal antibody asthe detection antibody (D), to detect BlaC antigen. C: G1 C: H1 C: 31AC: G1 C: H1 C: 31A 0.15 μg/mL 0.15 μg/mL 0.15 μg/mL .075 μg/mL .075μg/mL .075 μg/mL PBS 0.127 0.13 0.138 0.14 0.137 0.156 PBS 0.147 0.1290.124 0.143 0.194 0.161 BlaC 10 ng/mL 0.161 0.163 0.188 0.141 0.1370.131 BlaC 10 ng/mL 0.168 0.575 0.16 0.149 0.146 0.141 BlaC 20 ng/mL0.166 0.144 0.16 0.155 0.154 0.171 BlaC 20 ng/mL 0.211 0.148 0.165 0.1460.218 0.166 BlaC 50 ng/mL 0.182 0.318 0.172 0.232 0.193 0.142 BlaC 50ng/mL 0.529 0.167 0.18 0.168 0.171 0.144 BlaC 100 ng/mL 0.171 0.1650.174 0.154 0.178 0.207 BlaC 100 ng/mL 0.161 0.18 0.197 0.169 0.1640.149 BlaC 200 ng/mL 0.149 0.147 0.274 0.158 0.152 0.141 BlaC 200 ng/mL0.148 0.137 0.15 0.151 0.173 0.304 BlaC 500 ng/mL 0.144 0.188 0.1620.258 0.21 0.151 BlaC 500 ng/mL 0.166 0.199 0.165 0.176 0.165 0.15

Table 20 illustrates the effect Q column purification of rBlaC proteinon the ability of the Mouse anti-BlaC mAbs to pair with purified rabbitanti-BlaC polyclonal antibody to detect the rBlaC.

16) ELISA Optimization Assay: Purification of BlaC by Phenyl Sepharose

An optimization ELISA was performed where various amounts of BlaCantigen purified by phenyl sepharose, which is based on hydrophobicinteractions, were used to determine the effect of such isolation on theability for the Mouse anti-BlaC mAbs and partner reagents to detect therBlaC. The ELISAs used 0.15 μg/mL and 0.075 μg/mL of Mouse anti-BlaC mAb(from clones G1, H1, and 31A, as described above in part VI) as thecoating (“C” i.e., capture antibody) and 2.5 μg/mL purified rabbitanti-BlaC polyclonal antibody as the detection antibody (“D”). The bounddetection antibody was monitored using HRP-conjugated Ab at a titer of1:12,000 (rabbit). PBS and between 10 ng/mL and 500 ng/mL phenylsepharose-purified rBlaC were used as antigen.

The results of the ELISA optimization assay are set forth in Table 21.

TABLE 21 ELISA phenyl sepharose antigen purification on the ability ofMouse anti-BlaC mAb from clones G1, H1, and 31A as the capture antibody(C), paired with 2.5 μg/mL purified rabbit anti-BlaC polyclonal antibodyas the detection antibody (D), to detect BlaC antigen. C: G1 C: H1 C:31A C: G1 C: H1 C: 31A 0.15 μg/mL 0.15 μg/mL 0.15 μg/mL .075 μg/mL .075μg/mL .075 μg/mL PBS 0.153 0.142 0.179 0.139 0.15 0.121 PBS 0.14 0.1340.13 0.138 0.135 0.305 BlaC 10 ng/mL 0.188 0.164 0.208 0.176 0.18 0.168BlaC 10 ng/mL 0.159 0.143 0.205 0.159 0.167 0.141 BlaC 20 ng/mL 0.1560.166 0.144 0.172 0.134 0.156 BlaC 20 ng/mL 0.222 0.183 0.217 0.1620.152 0.186 BlaC 50 ng/mL 0.145 0.139 0.134 0.212 0.134 0.149 BlaC 50ng/mL 0.219 0.257 0.15 0.144 0.166 0.127 BlaC 100 ng/mL 0.164 0.1730.176 0.139 0.194 0.154 BlaC 100 ng/mL 0.193 0.218 0.189 0.144 0.160.128 BlaC 500 ng/mL 0.225 0.346 0.198 0.188 0.145 0.157 BlaC 500 ng/mL0.273 0.67 0.285 0.164 0.185 0.171

Table 21 illustrates the effect Q column purification of rBlaC proteinon the ability of the Mouse anti-BlaC mAbs to pair with purified rabbitanti-BlaC polyclonal antibody to detect the rBlaC.

Conclusion

The assays disclosed in this section demonstrate that the antibodyreagents generated as described above are useful for specificallydetecting BlaC, whether recombinant or wild-type, in biological samples.The assays can be designed with optimized concentrations and pairing ofreagents to promote high sensitivity and specificity, thus providingimproved methods and devices for detecting the presence of TB-complexbacteria.

While illustrative embodiments have been illustrated and described, itwill be appreciated that various changes can be made therein withoutdeparting from the spirit and scope of the invention.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A method of detectingthe presence of tuberculosis-complex bacteria in a biological sample,the method comprising contacting the sample with an antibody or antibodyfragment that specifically binds to a β-lactamase (BlaC) of atuberculosis-complex bacteria, and detecting the formation of a complexbetween the BlaC and the antibody or antibody fragment, wherein theformation of a complex is indicative of the presence oftuberculosis-complex bacteria in the sample.
 2. The method of claim 1,wherein the antibody or antibody fragment specifically binds to BlaCwith an amino acid sequence set forth in SEQ ID NO:2.
 3. The method ofclaim 1, wherein the antibody or antibody fragment comprises adetectable label.
 4. The method of claim 1, wherein the formation of acomplex between the BlaC and the antibody or antibody fragment isdetected by further contacting the complex with an affinity reagent thatcontains a detectable label and that specifically binds to the complex.5. The method of claim 1, further comprising contacting the biologicalsample with an immobilized BlaC protein or immobilized antibody orantibody fragment.
 6. The method of claim 1, wherein the biologicalsample is obtained from a subject suspected of havingtuberculosis-complex bacteria, and wherein the presence oftuberculosis-complex bacteria in the biological sample is indicative ofa tuberculosis infection in the subject.
 7. The method of claim 6,wherein the subject is human.
 8. The method of claim 1, wherein theantibody is a polyclonal antibody, a monoclonal antibody, a single chainantibody, an antigen binding enzymatic digestion product of theantibody, a chimeric antibody, or a humanized antibody.
 9. The method ofclaim 1, wherein the tuberculosis-complex bacteria are from one or moreof the species selected from the group consisting of: Mycobacteriumtuberculosis, Mycobacterium bovis, Mycobacterium bovis-BacillusCalmette-Guérin (BCG), Mycobacterium africanum, Mycobacterium microti,Mycobacterium canettii, Mycobacterium pinnipedii, and Mycobacteriummungi.